Higher resilience was associated with higher fruit  and vegetable  serves/day, more frequent breakfast consumption (p = 0.005), and less frequent soft drink (p < 0.001) and takeaway food consumption (p = 0.001). These results highlight a potential link between psychological distress and resilience with diet, and that further research in this area is warranted.University students report unhealthy diets and experience poorer mental health than the general population. This study explores the association between psychological distress and resilience with dietary intake in a sample of Australian university students. Cross-sectional data from the University of Newcastle Student Healthy Lifestyle Survey 2017 were analysed. Psychological distress , resilience  and fruit, vegetable, soft drink, takeaway food and breakfast intakes (short diet questions) were assessed. Socio-demographic , student  and health characteristics  were captured. Multivariate linear regression models explored associations between psychological distress and resilience with dietary intake, with adjustment for potential confounders. Analysis included 2710 students . In adjusted models, lower psychological distress was associated with higher fruit (β = −0.37,  University students’ mental health is a growing area of concern, with some evidence indicating a higher prevalence of mental health disorders among university students than in same-aged peers not at university and the general adult population ,2. The WThe high psychological distress rates among university students may stem from concern around academic factors (study time management and academic performance), as well as environmental factors  . The comPsychological distress among university students may also be associated with resilience; a measure of the ability to recover from stress . Cross-sIn the general adult population, observational studies consistently demonstrate that consuming an unhealthy diet is associated with a higher likelihood of mental health problems . Lower iMost studies exploring diet and mental health are in general adult populations or older adults, and of these, some indicate that the relationship between diet and mental health is moderated by other health behaviours or socio-demographic characteristics, for example physical activity, smoking, body mass index and age ,24,25. Fwww.surveymonkey.com.au) and was open from 4 September to 1 October 2017 (during the middle of the semester for most participants). The survey tool allowed access on a single device to prevent multiple entries by the same individual. The survey included 61 questions displayed over 27 pages and took approximately 15 minutes to complete based on pilot testing. Survey questions of a sensitive nature were optional to complete, including drug use, sexual health, and mental health questions. The current analysis specifically focused on mental health and dietary intake data. The study conduct and reporting were compliant with STROBE-nut guidelines [This study was a secondary analysis of data from an online cross-sectional survey, the 2017 University of Newcastle (UON) Student Healthy Lifestyle Survey (SHLS). The full survey methods and results have been published elsewhere . The aimidelines .n = 33,783) were invited to participate. Eligibility criteria included current enrolment as a student at the UON. To determine eligibility, a screening question asked individuals if they were a current student. The UON is a large urban university with students based at the main campus in Newcastle, New South Wales (NSW), Australia, additional smaller campuses across NSW (n = 4) and online/distance cohorts. Invitations were sent to students via an email to their UON email account on the 4th September 2017, with reminder emails sent on the 13th and 25th of September 2017. University teaching staff were emailed requesting they promote the survey in class or on the online learning management system using the recruitment materials provided by the researchers. The survey was also promoted via UON student social media accounts, digital signage across all campuses and posters at the main campus. On completing the survey, participants could choose to enter a prize draw to win one of five gift vouchers to the value of $AU100. All participants gave informed consent prior to participating. Study approval was obtained from the UON Human Research Ethics Committee (H-2015-0459).All students enrolled at the UON as of 4 September 2017 (“Don’t eat” to “≥6 serves”), where one serve was defined as 150 grams as per the Australian Guide to Healthy Eating (AGHE) [“Don’t eat” to “≥6 serves”), where one serve was defined as 75 grams [“≤1 cup/week” to “≥2 cups/day”), with 1 cup defined as 250 mL; takeaway meals or snacks (“Never/rarely” to “Everyday”); and breakfast (“Never/rarely” to “Everyday”). The questions assess usual intake, not a specific reference time period. The fruit and vegetable questions have demonstrated good relative validity and consistency compared with other dietary assessment methods; biomarkers and 24 h recall [Dietary intake was assessed using short diet questions from the NSW Adult Population Health Survey . This dig (AGHE) ; serves 75 grams ; consumph recall ,38. For h recall , where t(1 = None of the time to 5 = All of the time). The scores for each question sum to a total score between 10–50, which corresponds to the following categories of non-specific psychological distress; low (10–15), moderate (16–21), high (22–29), or very high (30–50) risk.Psychological distress was assessed using the Kessler Psychological Distress Scale (K-10) . The K-1(Strongly agree to Strongly disagree). Three of the items are negatively worded and three are positively worded, and responses are scored between 1–5, with a higher score indicating higher resilience. The participants’ average score across the items was calculated and compared with the following categories: low (1.00–2.99), normal (3.00–4.30) or high (4.31–5.00) resilience.Resilience was assessed using the Brief Resilience Scale (BRS) ; a measuSocio-demographic data collected included gender, age, country of birth, Aboriginal or Torres Strait Islander (ATSI) background, marital status, living situation and source(s) of financial support . Student specific data collected included degree type, faculty of study and number of years studying.2)) from participants reported height in cm and weight in kg, and compared with the World Health Organization BMI cut points [Physical activity (PA) was assessed using the Active Australia Survey, including the total time (minutes) and number of sessions spent walking and performing moderate and vigorous activity in the previous week . PA datat points .n = 114), resilience (n = 30), drug use (n = 35) or BMI (n = 26), or due to implausible data on physical activity (n = 160) or sleep (n = 1). Differences in demographic characteristics between those included in this analysis and those who chose not to complete the psychological distress and resilience questions were also assessed, with no significant differences found. Unadjusted linear regression models were used to explore associations between psychological distress  and resilience  with sociodemographic and student characteristics, health behaviours and dietary intake , to identify potential confounders. Statistical significance for the identification of potential confounders was set at p < 0.2. Fully adjusted linear regression models were used to explore associations between psychological distress and resilience with dietary intake, including the demographic and health characteristics found to be significant in the unadjusted models as potential confounders. Due to the large number of potential confounders identified and to prevent over-adjustment, backwards stepwise regression with comparison of Bayesian information criterion was used to eliminate potential confounders not contributing to the models and identify the models with best fit. Statistical significance for the fully adjusted models was set at p < 0.05. For measures with both a continuous and a categorical variable , the continuous variable was included in fully adjusted models.Analysis was conducted using STATA statistical software version 14.1. Demographic and health characteristics, dietary intake, psychological distress and resilience were described as percentages for categorical variables and means and standard deviations (SD) for continuous variables. A total of 3529 individuals responded to the survey, of which 3464 consented and were eligible to participate, and 3076 completed all compulsory questions. The total included in this analysis was 2710. Participants were excluded from the analysis if they were missing data on psychological distress (Participants’ mean ± SD age was 26.9 ± 9.5 years . The majp < 0.001).The percentage of participants in each category of non-specific psychological distress risk was 30.8% low, 32.0% moderate, 22.9% high and 14.3% very high risk. The percentage of participants with low resilience was 29.7%, with 59.9% classified as normal resilience, and 10.4% high resilience. The mean ± SD psychological distress risk score was 20.6 ± 7.8 out of 50, and the mean ± SD resilience score was 3.3 ± 0.8 out of 5. Unadjusted linear regression of psychological distress and resilience found that lower psychological distress score was significantly associated with higher resilience score  and fruit , higher frequency of breakfast consumption (p < 0.001), and lower frequencies of soft drink, cordial or sports drink (p < 0.001) and takeaway food (p < 0.001) consumption.In the adjusted models , controlp < 0.001) and fruit , higher frequency of breakfast consumption (p = 0.005), and lower frequencies of soft drink, cordial or sports drink (p < 0.001) and takeaway food (p = 0.001) consumption.In the adjusted models, higher resilience score  was significantly associated with higher serves per day of vegetables  . AdditioThere is a growing body of evidence to support the link between a healthy diet and better mental health outcomes ,49. MuchThe National Youth Health Foundation mental health service Headspace, is a mental health service aimed at early intervention for mental health related issues, with the most common intervention being cognitive behavioural therapy (CBT), and an average of four intervention sessions per individual ,55. AnalAnother interesting point of comparison is the magnitude of the association between mental health outcomes across the different dietary intake outcomes assessed. In the current study, larger estimates were found for the associations of soft drink, takeaway and breakfast with psychological distress and resilience than for fruit and vegetables. Similarly, Knowlden et al. reported a larger association between sugar-sweetened beverages and psychological distress than for fruit . In contAs with all cross-sectional surveys, the direction of the association between psychological distress, resilience and dietary intake cannot be determined from these data, nor causality. In addition, the use of short diet questions to assess dietary intake is limiting in terms of validity compared with other dietary assessment methods, such as validated food frequency questionnaire. However, this dietary assessment method was selected on the basis of the primary aim of the survey, i.e., monitoring and surveillance, and is still an appropriate method for this study. Further, a small number of dietary behaviours were assessed. It is also a limitation that body dissatisfaction and/or eating-disordered behaviour were not assessed in this study as associations have been found between these factors and dietary intake and mental health ,62,63. TThis cross-sectional analysis highlights a potential link between psychological distress and resilience with diet, however further research is needed to substantiate the findings. Future studies should include cohort studies to track changes in mental health and diet and their interrelationships prospectively. Further studies should also conduct more comprehensive evaluation of diet and using validated dietary assessment tools. In addition, it would be worthwhile to study the associations between diet and mental health in university students compared with matched non-studying peers. Longer term, the data collected from this and future studies of psychological distress, resilience and diet could be useful to inform mental health and/or dietary interventions to support university students.


Fallopia japonica on soil physical properties, microbial respiration, microbial biomass carbon content, enzymatic activities, and soil nematode communities. We established in total 30 plots in three natural habitats  that were either uninvaded or mostly monospecifically invaded by F. japonica. The soil physical and microbial properties differed among the investigated plots, but the differences were observed to be non-significant between the invaded and the uninvaded plots. Non-metric multidimensional scaling based on nematode species diversity indicated that the total number of identified nematode species and their abundance were higher in the uninvaded compare to the invaded plots. Negative effect of F. japonica on omnivores, plant parasites, and root-fungal feeder nematodes was confirmed by their lower abundance in the invaded compared to the uninvaded plots. In the invaded plots, we also confirmed lower Maturity and Channel index, but higher Enrichment index. Our results thus indicated that the invasive plant F. japonica could affect nematode communities, more than physical or microbial properties, regardless of habitat.This study determined the effect of the invasive plant  The impact of invasive plants on soil ecosystems in the last decades has attracted world-wide attention. Exotic plant invasions often have dramatic impacts on the resident vegetation by modifying its composition and structure . InvasivFallopia japonica (Houtt.) Ronse Decr., is considered to be one of the 100 worst invasive alien species in the world  to confirm or reverse the effect of F. japonica on selected food web components.Soil nematodes are an important group of soil biota, constituting an essential trophic link between primary decomposers, such as soil microflora, and larger animals and are recognized as useful bioindicators of soil conditions  due to thabitats . For thiF. japonica on the soil microbial and nematode community structure. Our objectives were to (i) determine the impact of F. japonica on soil pH and moisture, soil microbial respiration, soil microbial biomass carbon content, enzymatic activities and (ii) compare the abundance, diversity, trophic structure of nematode communities and selected ecological indices in three different habitats in F. japonica, the invaded and the uninvaded plots. The hypothesis tested was that soil physical properties, microbial, and nematode communities change when the ecosystem is disrupted by the invasion.To our knowledge, this is the first study observing the impact of F. japonica specimens appeared in the village of Opátka around 1992 . It was later probably transferred to the entire valley (7 km) below the village, invaded the banks of the creek and then spread to adjacent habitats thus creating large monocultures.The experiment was conducted in a valley near the village of Opátka in South Eastern Slovakia, Central Europe. This region has a temperate climate, with an annual average of 40 summer days per year and a warm, moderately dry sub-region with a mild winter. The average daily temperature in January ranges from 1.5 to 4.0°C, the average daily temperature in July ranges from 16.0 to 18.5°C, while the average annual temperature ranges from 5.0 to 7.0°C. The mean annual precipitation is 650 to 700 mm. The soils are characterized as Fluvisols, and the vegetation zone is characterized as Carpathian oak-hornbeam forest. The landscape is patchy, with deeply undulating uplands . The firF. japonica on soil physical properties, microbial, and soil nematode communities, we selected three habitats in the valley, namely, forest, grassland, and wetland and corresponding areas adjacent to them invaded by F. japonica.For studying the impact of Quercus robur, Q. cerris, Carpinus betulus, Acer campestre, and many shrub species such as mostly Viburnum sp. and Prunus spinosa.Forest (F) : covered by a natural, undisturbed, 100 years old deciduous Querco-Fagetea forest, mainly consisting of F. japonica (FF): a nearly monospecific stand of F. japonica covering an area of 500 m2, with an estimated time of invasion of 15 years.Forest edge invaded by Dactylis glomerata, Lolium perenne, Trifolium pratense, and Achillea millefolium; irregularly mown.Grassland (G) : covered with indigenous multispecies vegetation dominated by F. japonica (GF): an adjacent of F. japonica covering an area of 250 m2, with an estimated time of invasion of 15 years.Grassland edge invaded by Petasites hybridus, Caltha palustris, Galium aparine, Equisetum sp., Ranunculus sp., and Urtica sp.; the soil is regularly flooded mostly in the spring, and the vegetation is mown once a year in the autumn.Wetland (W) : covered by F. japonica (WF): an adjacent area of 200 m2, with an estimated time of invasion of 10 years, the soil is regularly flooded mostly in the spring.Wetland edge invaded by F. japonica. The distance between F, G, and W along the valley was approximately 1,500 m. A pair of the invaded and the uninvaded areas which did not differ in elevation, inclination, exposition, or management were chosen and the distance between the invaded and the uninvaded areas was 50 m. In each invaded area, we installed five randomly chosen 1 × 1 m plots (approx. 10 m apart) which had similar cover of F. japonica. Similar, five 1 × 1 m plots with random distribution were installed in corresponding uninvaded F, G, and W areas. This resulted in 30 plots . The uninvaded areas were assumed to represent the situation prior to the invasion of F. japonica.We selected a 25 m × 25 m area of the three different habitats  which was not yet colonized by 2), one from each corner and one from the center. The subsamples from each quadrat were then bulked to obtain five representative soil samples (1 kg) for each area. The soil samples were transferred to the laboratory in plastic bags. The bags were stored at 5°C until processing (storage time of soil samples were no longer than one week). Each sample was gently homogenized manually before processingThe soils were sampled using a garden trowel to depths of 0 to 20 cm in May 2016. A quadrat sampling method was used. Five soil subsamples were collected from each quadrat  in hermetically sealed bottles  in the extract was quantified by the oxidation with K2Cr2O7 dissolved in H2SO4 and titrimetrically by (NH4)2Fe(SO4)2. The same procedure was done with a non-irradiated sample (Cnon-irradiated). The microbial biomass carbon was then determined as MBC = (Cirradiated-Cnon-irradiated)/KME, whereby extraction efficiency factor KME = 0.213. The activities of acid and alkaline phosphatase were determined by the modified method of Soil microbial respiration (SMR) was measured by the amount of CO bottles  at 25°C Nematodes were isolated from 100 g of the mixed fresh soil samples by a combination of Cobb sieving and decanting  and a moHeterodera juveniles were extracted by floatation .The total number of species, total nematode abundance, mean number of nematodes per trophic group, and the Shannon and Weaver species diversity index (H’spp.)  were detThe differences in nematode characteristics  and basic ecological characteristics  were analyzed with two-way ANOVA with ‘ecosystems’ , ‘invasion status’ , and their interactions as factors. To meet the assumptions of these parametric tests, Box-Cox transformation was applied with the maximum likelihood approach and Golden Search iterative procedure on. If there was an interaction between ‘ecosystem’ and ‘invasion status’ , post hoc Fisher LSD test was applied separately for each ecosystem to determine the effect of ‘invasion status’. Otherwise, main factor ANOVA with two factors (without interaction) was applied. Consequently, in the case of confirmed significance of ‘ecosystem’, post hoc Fisher LSD test was used.As untransformed soil physical and microbial properties  were not normally distributed (Shapiro–Wilk test) and transformation did not improve normality, nonparametric statistics were applied. Differences among six combinations of ‘ecosystem’ and ‘invasion status’ were tested separately with Kruskal–Wallis ANOVA, followed by a post hoc multiple comparisons.p<0.05, 0.01, and 0.001.The above mentioned statistical analysis were performed using Statistica Cz, version 12.0  and signRelationships between plots, nematodes, and selected environmental characteristics  were analyzed by ordination techniques. Redundancy analysis (RDA) was performed using Canoco 5 , becauseNon-metric multidimensional scaling (NMS) ordination was used to examine any changes in the structure of nematode community for the invaded and the uninvaded habitats. A three-dimensional solution was executed by Autopilot, with the slow and thorough mode and Sørensen (Bray-Curtis) distance (recommended for community data). PC-ORD  was usedp < 0.001) among the investigated plots (Table p < 0.05) higher soil moisture only in the invaded FF than the adjacent uninvaded F. The pH varied from 5.2 in F to 6.8 in W and from 6.4 in FF to 7.2 in WF, but no significant differences were observed between the invaded and the uninvaded plots.The soil physical properties, soil moisture, and pH differed substantially  than in the uninvaded plots   in all plots with W) Table . OppositAcrobeles cylindricus, Eucephalobus oxyuroides, Mesorhabditis sp. juv., Plectus tenuis, Prismatolaimus dolichurus, and Punctodora sp. juv.; one fungivore Tylencholaimus stecki; one omnivore Aporcelaimellus obtusicaudatus; two predators Coomansus parvus and Thonus ettersbergensis; two plant parasites Heterodera hordecalis and Meloidogyne hapla; and two root-fungal feeders Boleodorus thylactus and Coslenchus costatus. On the other hand, only the invaded plots  contained the omnivore Axonchium coronatum (GF), the predators Coomansus zschokkei (GF) and Trischistoma monohystera (WF), and the plant parasites Hemicycliophora typica (WF) and Heterodera sp. 1 (FF) (Table p < 0.001). Subsequent post hoc Fisher LSD test confirmed a significant effect of ‘invasion status’ for F compare to FF and for W compare to WF (both p<0.05), but not for G compare to GF. Species diversity index did not differ significantly between the invaded and the uninvaded plots  than in the invaded plots (49 and 40). The species found in the uninvaded  but absent in the invaded plots were as follows: six bacterivores ts Table .F = 1.7, p = 0.01). Soil acidity and SMR were negatively correlated with the occurrence and abundance of most nematode species in F, G, and W, but soil pH positively correlated with the abundance of the predators A. coronatum and C. zschokkei in GF. The RDA partly separated the nematode communities of the uninvaded habitats from the invaded habitats. Cephalobus persegnis, Plectus parietinus, Prismatolaimus intermedius, and Mesodorylaimus bastiani were more abundant in the uninvaded plots, but Microdorylaimus parvus, Anatonchus tridentatus, and Rotylenchus robustus were more abundant in the invaded plots  that accounted for 53.5% of the total variation of nematode species abundance Fig. . Monte Cots Fig. .p < 0.0001) after 48 iterations, which was supported by a Monte Carlo permutation tests with a significance of p = 0.004 and a mean stress of 10.67 for real data and 250 runs for both real and randomised data. The variances explained by the first and second axes were 48 and 28%, respectively. The NMS analysis identified a notable impact of the invasive plants , 15 were plant parasites (24.2%), 9 were omnivores (14.5%), 7 were predators (11.3%), 6 were fungivores and root-fungal feeders (both 9.7%), and 1 was an insect parasite (1.6%).p < 0.01). Bacterivores were the second most abundant trophic group in F and G and the most abundant trophic group in W. Subsequent post hoc Fisher LSD test confirmed a significantly higher bacterivore abundance (p < 0.05) in F compared to FF and in W relative to WF (p < 0.05), but not for G and GF was observed. The abundance of fungivores was very low in the forest compared to both the grassland and the wetland (p < 0.001), but without significant differences between the invaded and the uninvaded plots. The abundance of root-fungal feeders was significantly higher in the forest and grassland rather than the wetland (p < 0.05) and in the uninvaded than the invaded plots (p < 0.01). The abundance of omnivores was significantly higher in the uninvaded rather than the invaded plots (p < 0.001). No significant differences in the abundance of predators were found.Plant parasites were the most abundant trophic group in F and G Table . The abup < 0.05). Structure and Basal indices did not significantly differ between the invaded and the uninvaded plots. When plotting the Enrichment and Structure Indices, most of the soil samples ended up in Quadrat B for both the invaded and the uninvaded plots (data not shown) suggesting that food webs were highly enriched and structured with both bacterial and fungal decomposition channels and maturing food web condition. No significant effect of ‘ecosystem’ was observed in the case of abovementioned nematode indices  in all ecosystems invaded by F. japonica in comparison to the uninvaded ones, suggesting that invasion did not affect microbial activity. This was in line with the measured enzyme activities, which remained unaffected by F. japonica invasion probably due the unchanged soil acidity, a factor affecting activity of soil enzymes . It may be that their diverse and often unknown feeding strategies is hampering data interpretation  indicates decomposition pathways dominated by bacteria whereas high CI (>50%) indicates a higher proportion of fungal decomposition  in all habitats. Surprisingly, F. japonica invasion negatively affected neither soil moisture nor soil acidity, and neither SMR and MBC content nor soil enzymes activity. But, there is some doubt that the abundant litter produced and the deep rhizome system of invasive F. japonica have an overall negative impact on soil moisture and pH to be the benefit of the invader, supported by our findings. Most of the studies performed were, however, local, including ours from three habitats. The extent of impact F. japonica invasion has on soil nematode communities, as well as microbial characteristics on the regional to national scale under a variety of natural conditions remains to be verified in future studies.In conclusion, our study demonstrated that the invasive plant species


Candida species to traditional drugs represents a great challenge to the medical field for the treatment of skin infections. Essential oils were recently proposed to increase drug effectiveness. Herein, we developed and optimized  Mediterranean essential oil  lipid nanoparticles for clotrimazole delivery, exploring the potential synergistic effects against Candida spp. Small sized nanoparticles (<100 nm) with a very broad size distribution (PDI < 0.15) and long-term stability were successfully prepared. Results of the in vitro biosafety on HaCaT  and A431 , allowed us to select Lavandula and Rosmarinus as anti-proliferative agents with the potential to be used as co-adjuvants in the treatment of non-tumoral proliferative dermal diseases. Results of calorimetric studies on biomembrane models, confirmed the potential antimicrobial activity of the selected oils due to their interaction with membrane permeabilization. Nanoparticles provided a prolonged in vitro release of clotrimazole. In vitro studies against Candida albicans, Candida krusei and Candida parapsilosis, showed an increase of the antifungal activity of clotrimazole-loaded nanoparticles prepared with Lavandula or Rosmarinus, thus confirming nanostructured lipid carriers (NLC) containing Mediterranean essential oils represent a promising strategy to improve drug effectiveness against topical candidiasis.The increasing development of resistance of  Candida species, thus causing severe opportunistic infections in immuno-compromised patients, especially those affected by cancer, diabetes and AIDS [Candida infections requires the use of azoles , polyenes  and echinocandins [Candida species are developing resistant mechanisms to the treatment with these drugs [Caryophyllata EO in SLN improved the rate of microbial killing, reducing the concentration for inhibiting and killing micro-organisms, demonstrating the advantages and efficiency of SLN in combating microbial pathogenesis [Zataria multiflora EO were successfully prepared, having a mean size of 650 nm, with 93.2% of the essential oil released after 24 h [Eucalyptus EO-loaded nanostructured lipid carriers NLC were developed exploiting a synergistic effect between the EO and oleic acid, whose synergism enhanced cell proliferation to confer an advantage in the treatment of wound healing [Artemisia arborescens was successfully incorporated into SLN without affecting the in vitro EO antiherpetic activity [Artemisia arborescens oil accumulation into the skin, and that oil permeation occurred only when the oil was delivered from the control solution [Rosmarinus officinalis, Lavandula x intermedia “Sumian” and Origanum vulgare subsp. hirtum can be successfully used as matrix components and active ingredients of nanostructured lipid carriers (NLC), thus exploiting their relevant anti-inflammatory (or antioxidant) activity, enhancing their biocompatibility and reducing the cytotoxicity of the pure oils [Recently, the worldwide incidence of serious systemic infections has extremely increased, most of these due to fungi of and AIDS . Nowadayocandins ,3. Howevse drugs ,4. In adse drugs ,6. Basedse drugs ,8. It hase drugs . EOs phase drugs ,11,12. Nse drugs ,14. In ose drugs ,17,18,19ogenesis . SLN conure oils . Other aure oils , chitosaure oils ,21.Rosmarinus officinalis, Lavandula x intermedia “Sumian” and Origanum vulgare subsp. hirtum as intrinsic and active oily liquid component for the optimization of NLC by design of experiment (DOE). The quality by design (QbD) was developed focusing on the quali-quantitative surfactants mixture, for all the selected EOs. NLC were characterized in terms of mean nanoparticles size, polydispersity, structure and long-term physical stability exploiting LUMiSizer® technology. In order to deepen the cytotoxicity effects of EOs on microorganism we also performed calorimetric study on biomembrane models. Furthermore, we aimed to evaluate the biosafety of the selected EOs, pure and loaded into NLC, and to compare the biological effects on two cell lines, namely HaCaT  and A431  selected on the basis of the potential topical application. In vitro antifungal activity against three reference strains of Candida spp., namely C. albicans, C. krusei and C. parapsilosis was evaluated.Based on these considerations, the aim of the present work was to develop and optimize Mediterranean EOs-loaded NLC for the combined delivery of clotrimazole (CLZ) thus exploiting their potential synergistic effects to the benefit of resistant topical candidiasis treatment. Based on our recent findings, we selected Rosmarinus officinalis L., Lavandula x intermedia “Sumian” and Origanum vulgare subsp. hirtum essential oils were kindly provided by Exentiae s.r.l. . All solvents were for chromatography (LC grade) and were bought from VWR International . 1,2-Dimyristoyl-sn-glicero-3-phoshocholine (DMPC) was a gift from AVG s.r.l. . Culture media  and RPMI 1640, fetal bovine serum (FBS), l-glutamine, sodium pyruvate, Versene, trypsin (0.05% trypsin-EDTA), and antibiotics (streptomycin and penicillin) were purchased from Gibco . Alamar Blue® reagent was purchased from Invitrogen Life-Technologies . Potato dextrose agar (PDA) medium (Sigma) was purchased from Laborspirit Lda. .Kolliphor RH40 was kindly provided by BASF Italia S.p.a.  while Oleoyl Macrogol-6 Glycerides (Labrafil) was a gift from Gattefossé Italia s.r.l. . Hydrogenated Coco-Glycerides (Softisan 100) was purchased from IOI Oleo GmbH . Polysorbate 80 (Tween 80), trehalose and chloroform were purchased from Sigma Aldrich Co. . Lavandula (LNLC), Origanum (ONLC) and Rosmarinus (RNLC) EOs were produced by high-pressure homogenization (HPH) using the Ultra-Turrax® , as previously reported [o/w) nanoemulsion was further processed using a high pressure homogenizer  at 70 °C for three cycles. The final formulation was then cooled to room temperature leading to the lipid phase recrystallization and finally the lipid nanoparticles were formed [Lavandula (CLZ-LNLC) and Rosmarinus (CLZ-RNLC), were prepared adding CLZ (0.5% w/v) to the lipid phase during the preparation procedure.NLC with reported . For alle formed ,22. Drug3 full factorial planning). The design was highly suitable for the investigation of quadratic response surface and for generating a second order polynomial model, applied to describe the principal effects and interaction among the identified variables. The independent variables studied in this design such as the concentration of surfactant (A) and the amount of co-surfactant (B) were investigated at three different levels  and were represented by (−1), (0) and (+1): (i) Surfactant: 3% w/v (−1), 4.35% w/v (0), 6% w/v (+1); (ii) Co-surfactant: 1.5% w/v (−1), 2.2% w/v (0), 3% w/v (+1). ANOVA test was applied to verify the fitted model. Statistical analysis was considered significant when the p values were less than 0.05.In order to optimize NLC composition, an experimental design was performed by using StatSoft7. A full factorial design was employed for the study, based on two factors and three levels (2Mean particle size (Zave), polydispersity index (PDI) and zeta potential values (ZP) of all unloaded and CLZ-loaded NLC were determined by Dynamic Light Scattering (DLS) using a Zetasizer Nano S90 . For measurements, samples were properly diluted (50 µL) in 1 mL of ultra-purified water. Each value was measured at least in triplicate and results are shown as mean ± standard deviation (SD).The amount of EO in the NLC was determined as encapsulation efficiency (EE%) by UV spectroscopy , using av/v)), penicillin (100 U/mL), streptomycin (100 µg/mL) and L-glutamine (1 mM), in an atmosphere of 5% CO2/95% air, with controlled humidity, at 37 °C. Cells were grown, in T25 culture flasks, to near confluence and then were subjected to the action of trypsin, counted  and the required volume of cells was re-suspended in FBS-free culture media to give a final density of 5 × 104 cells/mL. Cells were then seeded into 96-well microplates , maintained in incubator for 24 h (for adherence), for other details see [Cell culture, maintenance and manipulation: HaCaT , Eppelheim, Germany)  and A431ails see ,24.® method. A stock solution of each EO was prepared by diluting each one with DMSO (1:1 v/v). Before the cell treatment, test solutions were obtained by diluting the stock solution in FBS-free culture media, to give concentrations ranging from 0.001% (v/v) to 0.5% EO (v/v). In order to analyze the cell viability of NLC, each formulation was diluted in FBS-free culture media oils. Cells were treated with different concentrations of the pure EOs and NLC for 24 h. After this period, samples were removed and Alamar Blue solution (10% (v/v) in FBS-free medium) was added (100 µL/well). After 5 h of incubation, the absorbance was read at 570 and 620 nm using a Multiskan EX microplate reader , and the cell viability was calculated using the equations recommended by Alamar Blue manufacturers, as described in [Cytotoxicity assay: The cytotoxicity of all samples (pure EOs and NLC systems) was evaluated through the Alamar Blue−1 were obtained from a wave number 650–4000 cm−1, using a speed of 0.50 cm/s and a force gauge of 100 [Freeze-dried NLC obtained with the addition of trehalose (NLC:cryoprotectant ratio 1:10), freezing suspensions at −80 °C and lyophilizing in a freeze-dryer  for 48 h were analysed by Fourier transform-infrared (FT-IR). FT-IR characterizations of pure CLZ and freeze-dried CLZ-loaded NLC were prepared by using different essential oils were performed using a FT-IR spectrophotometer  equipped with an ATR accessory of diamond Zn/Se. For each sample, 16 scans at a resolution of 2 cme of 100 .® . The instrument employs the patented STEP®-Technology, which permits the obtainment of Space- and Time-resolves Extinction Profiles, thus measuring the intensity of the transmitted light during centrifugation, as a function of time and position, over the entire sample length. An accurate highly accelerated qualitative description of the colloidal suspensions stability was performed by means of the multisample analytical centrifuge LUMiSizerg is the gravitational acceleration constant, ρp and ρ are respectively the densities of the particle and fluid, dp is the particle size and η is the fluid viscosity. As previously reported [Parallel near infrared light illuminated the entire sample cell and the transmitted light was detected by sensors arranged linearly across the sample from top to bottom. Transmission was converted into extinction by log I/I0 and particle concentration could be calculated. The progression of the transmission profiles contained the complete information on the kinetics of any concentration changes due to creaming, sedimentation, flocculation, coalescence or phase separation . In addireported , the cloThe amount of the encapsulated CLZ was determined after ultracentrifugation, pellet disruption in Tetrahydrofuran anhydrous (THF), vortex and UV spectrophotometry . The encapsulation efficiency (EE%) was calculated by the ratio between the amount of drug entrapped in the nanoparticles and the total amount of drug used for their preparation (Equation (1)). The loading capacity (LC%) was calculated by the ratio between the amount of drug unencapsulated in the nanoparticles and the total amount of lipid used for their preparation (Equation (3)):v/v) for ensuring pseudo-sink conditions. The receiving solution was constantly stirred and thermostated at 35 °C to maintain the membrane surface at 32 °C. 500 µL of each formulation was applied in the donor compartment on the membrane surface under non occlusion conditions and the experiments were run for 7 days. At fixed time intervals, 200 µL of the receptor phase were withdrawn and replaced with the same volume of receiving solution. Samples were analysed spectrophotometrically to determine CLZ content. CLZ release from NLC was evaluated by Franz-type diffusion cells . Before the experiment, the cellulose membranes were moistened by immersion in water for 1 h at room temperature before being mounted in Franz-type diffusion cells. The receptor was filled with water/ethanol  on multilamellar vesicles (MLV), different volumes  of each EO and PBS  were added to the prepared MLV to obtain 1 mL as final volume. Afterwards, each sample was diluted 1:10 with PBS. The final concentration of the EO in the obtained MLV was in the range of 0.2–0.01% (v/v). DSC analyses were performed on these final diluted samples. Biomembrane models were prepared using the TLE method. Briefly, DMPC (25 mg) was dissolved in chloroform in a Pyrex glass test-tube. The organic solvent was removed at 30 °C on nitrogen stream rotavapor . The formed phospholipid films were dried using a Büchi spray dryer (Büchi TO-51) for 24 h, then hydrated by adding different volumes  of isotonic PBS pH 7.4. The tube was alternatively vortexed  and warmed in a water bath at 40 °C for 3 min twice. The temperature was kept higher than that of DMPC gel-liquid crystal phase transition (24 °C) thus allowing the complete hydration of the phospholipids. In order to evaluate the influence of the two selected EOs  was used to perform calorimetric analysis. The detection system was an HSS8 high sensitivity sensor  and a ceramic sensor  with 56 thermocouples. The signal time constant was 18 s and the digital resolution of the measurement signal was less than 0.04 μW. Calorimetric resolution and sensitivity determined by TAWN test were respectively 0.12 and 11.9. The sampling rate was 50 values/s. The sensitivity was automatically chosen as the maximum possible by the calorimetric system, and the reference was an empty pan. The calorimetric system was calibrated, in temperature and enthalpy changes, by using indium by following the procedure of the DSC 1 Mettler TA STARe instrument. The reference was an aluminium pan containing 100 μL isotonic PBS (pH 7.4). Each sample was submitted to heating and cooling cycles (two times), in the temperature range 5–55 °C, at a scanning rate of 2 °C/min for heating and 5 °C/min for cooling. We evaluated the results of the second heating. Thermotropic parameters were calculated with Mettler STARe Evaluation software system (version 15.01) installed on an Optiplex 3020 DELL. Pure EOs and NLC were studied with DSC in the same range of temperature used for MLV investigations, results are reported in Candida spp., namely C. albicans ATCC 10231, C. krusei ATCC 6258 and C. parapsilosis ATCC 90098. For comparative purposes, in addition to the CLZ-LNLC and CLZ-RNLC formulations, LNLC and RNLC systems were also tested, as well as free EOs (L and R) and free CLZ. Briefly, inoculum suspensions were prepared at appropriate densities in RPMI 1640 broth  from 24h PDA cultures. Stock solutions of free EOs, CLZ and NLC formulations were prepared in MilliQ water using Tween 80 as a co-solvent, sterilized by filtration and then diluted in RPMI 1640 broth. Free EOs and all NLC formulations were tested at different concentrations in the range 0.03125–2% v/v while free CLZ was tested in the range 2–128 µg mL−1, determined based on the combined information available in KnowledgeBase (http://antibiotics.toku-e.com) for the three strains. NLC-free growth controls, sterility and Tween 80 wells, were also included. The microplates were incubated for 48 h at 35 °C.The in vitro antifungal susceptibility test for the selected CLZ-NLC formulations was performed based on the Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3 broth microdilution (BMD) method  for yeasAfter incubation, the yeast growth was screened by OD measurement at 525 nm and the content of first five dilutions tested of each formulation was transferred into to PDA plates and incubated for a new 24 h period at 35 °C. Minimum inhibitory concentration (MIC) values, considered as the lowest concentration of each solution causing full growth inhibition, and minimum lethal concentration (MLC) values, considered as the lowest concentration of each solution causing fungal death, were determined after visual counting of yeast colonies.p < 0.05 was considered statistically significant. Significant differences (p < 0.05) between EO and respective NLCs in cell viability results for the two cell lines were obtained by using two-way ANOVA with a Tukey post-hoc test. All plotted data were presented as a mean of three different experiments ± SD. Differences between the calculated means of each individual group were determined by one-way ANOVA test using the statistical tools available in GraphPad Prism 6 software. A value of In this work, we exploited the Design of Experiments (DoE) as a tool of the Quality by Design approach (QbD), with the aim to develop optimized formulations in terms of particles with small size and high homogeneity. In particular, a full factorial design was developed with two independent variables  and two levels  .As previously reported in literature, the QbD led to a huge amount of information, saving both time and costs of the experiment ,30.Lavandula x intermedia “Sumian” showed that nanoparticle features are affected by the amount of surfactant and cosurfactant. In particular, a wide variation in response was obtained, with Zave values ranging from 70 nm to 200 nm, while PDI ranged from a minimum of 0.11 to a maximum of 0.35. Coefficient regression values were found to be higher for Zave when compared to PDI. The highest coefficient value found for the surfactant (β1) demonstrates a very high significant effect  nanoparticles homogeneity, as shown in It is worthy to note that, surprisingly, by decreasing the amount of co-surfactant, an insignificant effect was observed on mean particle diameter. Thus, the exerted energy during nanoparticle production would be enough for coating nanoparticles’ surface, even using less co-surfactant, thus resulting, contrary to previous literature findings, in reduced sized nanoparticles ,33,34,35Rosmarinus and Origanum (data not reported).Similar findings were observed in the response surface plot obtained for the second order polynomial equation of NLC prepared with The above considerations highlight that the amount of the surfactant mixture was able to significantly affect the physicochemical properties of EOs NLC but not the EE% of the EO, that was very high (~96%) for all EOs, in accordance with previous findings ,20. FurtLavandula EO, has been reported to have positive effects in the treatment of eczema, dermatitis, in the improvement of skin wound healing with less scars and has been recommended for the treatment of skin diseases such as psoriasis [The beneficial effects of EOs on dermatological applications have been established for a long time, although most available information comes from ethnopharmacological surveys. As example, the use of soriasis . Lavandula, Origanum and Rosmarinus) as well as of the respective prepared NLC , and to compare their biological effects on two cell lines, namely HaCaT  and A431 . For that, HaCaT and A431 cells were exposed to a set of concentrations of EOs (see methods for details) during 24 h, and cell viability was accessed using the metabolic indicator Alamar Blue. HaCaT and A431 cell viability results (expressed as percentage of control (non-exposed cells)) of the treatment with the pure EOs are reported in In this study we aimed to evaluate the biosafety of the three EOs of the Lamiaceae Family  and 0.274 ± 0.006 (% v/v) which are ~10-fold (HaCaT) and ~23-fold higher than those obtained for Origanum  showed IC50 values of about 0.0027% (v/v) for all cells [Origanum pure EO cytotoxic effect was higher  in the treatment against the tumoral cell line compared to HaCaT, a normal keratinocytes cell line  for HaCaT and about 0.114–0.117% (v/v) for A431 cells  . Herein Lavandula and Rosmarinus (50 ng/mL) may be regarded as anti-proliferative agents and have potential to be used as co-adjuvants in the treatment of non-tumoral proliferative dermal diseases, such as infections, psoriasis, eczema, ichthyosis.Based on the presented data, NLC prepared with Lavandula and Rosmarinus were selected for CLZ delivery. As reported in Lavandula EO was shown in respect to RNLC. However, the low values found for the EE% could be due to steric hindrance imparted to CLZ because of the spatial orientation of the phenyl rings, thus limiting not only the in vitro performance as reported by Sabzevari et al., but also the interactions with other components during the formation of NLC [−1, corresponding to the benzene ring stretches; peaks at 906.2, 821.6 and 756.3 cm−1 correspond to C–H stretches; bands at 1082.0 and 1206.2 cm−1 corresponding to chlorobenzene and C–N stretching, respectively , 1728, 1739 cm−1 (carbonyl compound CHO), 1000–1300 cm−1 (C–O stretching), as we previously reported [Lavandula and Rosmarinus EOs characteristic bands were in the range 3200–3600 cm−1 (O–H stretch), range 3000–3100 cm−1 (C–H stretching), 1670–1820 cm−1 (C=O), range 1400–1450 cm−1 (bending vibrations of CH2 and CH3 groups), range 1000–1330 cm−1 (C–O–C) and range 675–1000 cm−1 (C–H bending) [Lavandula and Rosmarinus  . Thus, Fsmarinus .®. The innovative technology offers the possibility to achieve more time-saving information on sample stability (in terms of particles separation) compared to the traditional visual observation tests that take extensive period to determine the long-term stability of a colloidal suspension. LUMiSizer® is able to detect fast stability ranking and shelf-life estimation of undiluted dispersions at their original concentration, in minutes/hours instead of months/years, exploiting centrifugal force to accelerate the occurrence of instability phenomena  [Lavandula and Rosmarinus EO was evaluated measuring the nanoparticles migration phenomena at high RCF, thus obtaining the creaming velocity by tracking the front movements at a specific transmittance with time  . Consideith time . As repo systems ,45. Thus® at high RCF with the theoretical model (Stoke’s law), as previously reported by Chang et al. [In order to achieve more information on CLZ-RNLC long-term stability, we compared results of the creaming velocity obtained with LUMiSizerg et al. . In FiguThe results obtained by the accelerated stability studies, allowed us to establish the potential shelf life of the prepared NLC colloidal suspension, according to the highest industry norms and regulations ISO 13318-1 and ISO 13318-2 (concerning nanoparticle accelerated stability studies). In particular, since the nanoparticle separation, due to the occurrence of creaming phenomena, was not relevant for sample RNLC and CLZ-RNLC, we could reasonably estimate a shelf life of 12 months at 25 °C, at least. It is worth noting that the obtained stability results were in perfect agreement with PCS measurements of samples analyzed after 2 and 6 months of storage, showing the absence of significant variations in mean size and polydispersity values .Candida species [Lavandula or Rosmarinus EO as potential synergistic oily components. In vitro release profiles of CLZ from the prepared NLC are shown in As reported in literature, increasing resistant phenomena have been developed against  species ,4,46. InLavandula EO allowed the encapsulation of a greater amount of CLZ (45.81 mg) into the NLC compared to Rosmarinus (25.12 mg), it promoted a slower drug release compared to CLZ-RNLC. Indeed, after 24 h a similar amount of CLZ was released from both formulations , but after 120 h from the beginning of the experiment, less than 10% of CLZ was released from LNLC while RNLC released almost 40% of the encapsulated drug. These data demonstrate the possibility to prepare lipid nanoparticles with different release profiles, depending on the selected EO used as oily matrix component. In particular, the interesting behavior of CLZ-RNLC is not only related to its ability to provide a sustained prolonged CLZ release, but also to the possibility to economize the amount of the loaded drug, determining the formation of a nanosized colloidal suspension characterized by a long-term physical stability, as previously demonstrated.It is worth noting that even if Lavandula and Rosmarinus as pure EO were investigated for their potential capability to interact with model membranes through calorimetric technique. Each oil is characterized by a mixture of different components that influence biomembrane structure [In order to characterize the potential mechanisms related to cytotoxicity effects, tructure . It is kM rather than the enthalpy values of the main transition peak of DMPC bilayers  and the immediate lower concentration value allowed the growth of the inoculum (no increase on cell counting). Thus, we considered that the MIC and MLC values were the same for each formulation within the tested concentration ranges. The MICs and MLCs determined for C. albicans ATCC 10231, C. krusei ATCC 6258 and C. parapsilosis ATCC 90098 are summarized in Based on the encouraging results obtained testing EOs with model membranes, preliminary studies on the antifungal activity of CLZ-loaded EO-NLCs were performed, evaluating the susceptibility of three C. parapsilosis is the most sensitive strain to the majority of the tested solutions, while C. krusei seems to be the most resistant one among the three reference strains. Regarding the tested pure EOs (Lavandula and Rosmarinus), although little information is provided in the literature, the MICs and MLCs determined for each strain are in accordance to previous findings by Bona et al. [v/v) for both EOs in reference and clinical isolated strains of C. albicans. Interestingly, Lavandula as pure EO was found to be more active than Rosmarinus EO  compared to Rosmarinus , whose antifungal activity against several strains of Candida, including C. albicans, C. glabrata, and C. parapsilosis, have been previously reported [Rosmarinus antifungal activity, such as the lack of anti-candidal activity of R. officinalis L. samples collected from National Parquet El Hamma (Algiers) reported by Djeddi et al. [Lavandula EO of four-fold for C. albicans, two-fold for C. krusei and eight-fold for C. parapsilosis, while CLZ-RNLC provides an increase of the antifungal activity of the Rosmarinus EO of eight-fold for C. albicans, four-fold for C. krusei and four-fold for C. parapsilosis. Moreover, the antifungal activity of free CLZ is also increased when loaded into both EO-NLCs for C. albicans and C. parapsilosis.A primary overview at these results shows that a et al.  within trinus EO . This coreported . Howeveri et al. . Our fini et al. . In partLavandula and Rosmarinus, whose synergistic effects are to be further investigated, and could offer a strategy in using CLZ-EO-NLC for overcoming drug resistance mechanisms involved in the treatment of topical infections.Taken all together, our results allow us to infer that the nanoencapsulation of the selected antifungal drug into NLC systems prepared using Mediterranean EOs as intrinsic oily components represents a promising strategy to improve CLZ effectiveness against candidiasis. In particular, our results open the debate concerning the possibility to exploit the intrinsic properties of


This study was to analyze the outcomes of left subclavian artery (LSA) revascularization during thoracic endovascular repair (TEVAR) for type B aortic dissections (TBAD). From 2011 to 2017, TBAD patients who underwent LSA revascularization during TEVAR were enrolled. Technical success, endoleaks, mortality, complication, reintervention, and patency of target vessels were analyzed. 38 patients were included, 14 underwent carotid-subclavian bypass (CSB), and 24 underwent chimney graft (CG) implantation. Technical success rates were 92.9% and 100% in CSB and CG group. Eleven immediate type I endoleak (EL-I) was detected, including one from CSB group and ten from CG group. Three immediate type II endoleak (EL-II) was detected in CSB group. Perioperative complications showed no difference, but CSB group had longer intensive care unit (ICU) stay time. Median follow-up time was 26.2 months, and overall mortality was 14.3% and 0% in each group. Three EL-I and one EL-II underwent reintervention. All the LSA showed good patency, except one suffered from CG collapse. Both CSB and CG were feasible strategies to preserve the antegrade blood flow of LSA, and each strategy had its advantages and disadvantages. Based on our current experience, we preferred CG for high-risk patients. However, the evidence was still not strong enough, further well-designed studies are necessary to identify the criteria for LSA revascularization strategy during TEVAR. However, it had its limitations, one of which was the necessity of an adequate, disease-free proximal seal zones for the aortic stent graft. Anatomic challenges abounded for vascular surgeons because the lesions probably involving arch branches, usually left subclavian artery (LSA). A review of the literature suggested that 10–50% LSA needed to be covered intentionally to achieve an adequate seal zone3.For Stanford type B aortic dissection (TBAD), the thoracic endovascular aortic repair (TEVAR) had demonstrated favorable short- and mid-term results4.In 2009, Society for Vascular Surgery (SVS) published practice guidelines on the management of LSA during TEVAR, in which recommendations were made that the LSA should be revascularized before or after TEVAR as appropriate. However, the recommendations were based on low-quality evidence and did not address which revascularization strategy would be better during TEVAR for TBADIn the present study, we shared our experiences in LSA revascularization by carotid-subclavian bypass (CSB) and chimney graft (CG), and analyzed the outcomes of these two strategies during TEVAR for TBAD.From January 2011 to April 2017, the data of TBAD patients who underwent TEVAR with LSA revascularization were reviewed. All the patients underwent preoperative CTA scans to evaluate the extension of aortic dissection, measure the distance between LSA and primary entrance, and determine the intended proximal landing zone (LZ).At the initial stage, we preferred to CSB for LSA revascularization because of the sophisticated bypass technique. However, it was relatively traumatic and time-consuming. With the accumulation of experience, CG was preferred at the late stage.5. Except for emergent situations, CSBs were performed before the implantation of aortic stent grafts. The LSA was exposed through supraclavicular incision, PTFE vascular prostheses  were anastomosed side-to-side between the second section of LSA and left carotid artery, and occluders  were implanted to close the orifice of LSA to prevent type II endoleak (EL-II). Self-expandable covered stent grafts  were used in the CG group. After the aortic stent graft was deployed, CG was deployed rapidly parallel to it with at least 1 cm overlapping and slightly protruding proximally.All the TEVAR were performed under general anesthesia and with the use of intravenous heparin in a hybrid operation room or angiography suite. Valiant  aortic stent grafts were used in all patients and proximally deployed in zone 2 (Z2), as specified by IshimaruTechnical success, endoleaks, 30-day and overall mortality, perioperative complications, reintervention, and patency of target vessels were analyzed. Completion ascending aortic angiogram was performed to evaluate the immediate results. Technical success was defined as the instant postoperative aortic angiogram demonstrating successful implantation of the aortic stent graft, the lesion was excluded, and the LSA had favorable antegrade blood flow, no large amount of type I endoleak which needed to be converted to open surgery or implant another aortic stent graft proximally.Follow-up examinations included CTA scan and ultrasonography, performed at postoperative 1, 3, 6, 12 months and then yearly thereafter. Symptoms and clinical signs were also collected during clinic visits.2 probability test in a 2 × 2 table. A P-value of <0.05 was considered statistically significant. This retrospective study had followed the principle of the Declaration of Helsinki, the West China Hospital Ethics Committee had approved this study, and patient consent was waived.SPSS 24.0 was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation. The events were calculated with frequencies displayed as counts and percentages and analyzed by Fisher exact or χA total of 38 patients  were included, their characteristics were listed in Table The technical success rates were 92.9% (13/14) and 100% (24/24) in CSB and CG group, respectively. One perioperative death occurred in CSB group due to severe acute renal injury. The length and diameter of proximal LZ were 17.9 ± 4.3 mm and 31.9 ± 4.2 mm respectively. A total of 51 aortic stent grafts were used. The diameter of aortic stent graft was 32.4 ± 3.9 mm (range 24–42 mm), the covered length was 215.8 ± 30.2 mm (range 150–280 mm). The diameter and length of CGs were 9.4 ± 1.1 mm (range 8–12 mm) and 56.7 ± 9.6 mm (range 40 to 80 mm). All the prostheses used in CSB were 6 mm in diameter.Immediate EL-I was detected in 11 cases 11/38, 28.9%) on postoperative aortogram, including 1 from CSB group and 10 from CG group. These EL-Is were small amount, and did not need to be converted to open surgery or implant another aortic stent graft proximally. All of the CGs were balloon dilated at the same stage, 5 EL-I were mildly alleviated. Immediate EL-II was detected only in the three emergent patients from CSB group, who did not undergo LSA occlusion. Other perioperative complications included spinal cord ischemia (n = 2), pulmonary infection (n = 9), and incision complications (n = 5). The incidence rates of stroke and retrograde type A dissection were 0%. The hospital and intensive care unit (ICU) stays were 17.1 ± 7.6 days and 4.6 ± 5.9 days, respectively. Table 8.9% on pAll of the 37 patients were successfully followed up with a median period of 26.2 months. During follow-up, one patient from CSB group died of respiratory failure two months after the operation. All the patients showed good LSA patency, except one suffered from CG collapse Fig. . HoweverThe outcomes during follow-up were listed in Table 2, and LSA coverage was necessary to achieve good proximal fixation for those with an insufficient proximal landing zone. However, intentional coverage of LSA might not be physiologically tolerated because the LSA provides extensive circulation to the left upper limb, spinal cord and posterior cerebral circulation. A few of studies had reported their clinical outcomes on LSA revascularization during TEVAR, most of them only reported CSB or CG, no comparison had been made between these two strategies. We evaluated the available data regarding CSB and CG in TEVAR and summarized our experience with them.TEVAR had shown favorable short- and mid-term result for patients with Stanford type B aortic dissection6. In the present study, no patient suffered from stroke perioperatively or during follow-up. Feezor et al.3 and Zamor et al.7 had reported that CSB could reduce the risk of stroke in TEVAR. And the reported stroke rate after CG implantation was lower than LSA coverage during TEVAR11. So both CSB and CG showed good effects on preventing stroke.Stroke is a major complication of LSA coverage in TEVAR. According to a meta-analysis on the morbidity and mortality during zone 2 TEVAR, LSA coverage was associated with an increased risk of stroke12. Risk factors for SCI had been reported previously including the length of aortic coverage, prior abdominal aortic aneurysm repair, hypotension, and LSA coverage15. For patients with both extended aortic coverage and LSA coverage, LSA revascularization was necessary to maintain the perfusion of the spinal cord. In the present study, 18 cases had aortic coverage more than 240 mm in length, and 2 of them had postoperative neurological deficits  referable to SCI. After cerebrospinal drainage and systemic blood pressure elevation, their symptoms were resolved. As reported16, hemodynamic control, cerebrospinal drainage, and neuroprotective drugs should be used during TEVAR for the patients associated with risk factors for SCI.Spinal cord ischemia (SCI) is another devastating complication of LSA coverage in TEVARet al.17, the patency rate of CSB in TEVAR was 95.2% (20/21) at a 74.6 months follow up. Similarly, Xue et al.18 reported that 91.1% (51/56) patients showed good CG patency during 16.5 months follow up. Besides, a multicenter retrospective study also reported a 98% primary patency of the CG9. So we believe both CSB and CG were effective method to maintain the antegrade blood flow of LSA.Patency was important for LSA revascularization. In the present study, all of the CSB prostheses and more than 95% CGs maintained patent during follow up. As reported by Martin 20, the radial support force was too strong in aortic stent graft or too weak in CG could both increase the risk of chimney stent compression or occlusion. Besides, in a study reported by Pecoraro et al.21, incomplete CG expansion, inadequate length and use in small and diseased target arteries were risk factors for occlusion.Although it is rare, one CG was compressed and collapsed in our study, but no reintervention was needed. A few studies had reported the risk factors of CG compression. Some researchers had reported that CG compression was device related22. The immediate EL-I rate was significantly higher in CG group , and there were still five EL-I remained during follow-up, which led to reinterventions for false lumen enlargement and aortic stent graft immigrant. In a systematic review conducted in 201523, the overall early EL-I rate of thoracic CGs was 11%, and 42% ELs-I were treated by embolization, extension of aortic stent graft or even open conversion. Recent studies26 also reported a similar result. So we believed that CG brought more EL-I and had a higher chance of reintervention.Endoleak was another concern of LSA revascularization. The CG was expected to prevent endoleaks by prolonging the channel existed between CG and aortic stent graft. However, it was reported that the deployment of CG might potentially displace the aortic stent graft and induce an endoleakConcerning the indication of CSB and CG in TEVAR, they can be both applied in the cases requiring LSA revascularization, especially for those with dominant left vertebral artery or left upper extremity dialysis access. However, no criteria had been reported that which method should be chosen for LSA revascularization. There was no doubt that CSB was more traumatic than CG, which could result in longer operation time and more complicated postoperative course. In the present study, the ICU stay time was significantly longer in CSB group than CG group, and the pulmonary infection rate was higher in the bypass group (42.9% vs. 12.5%). Besides, the events of CSB group concentrated in the earlier than CG group, so we preferred CG for high-risk patients  for a stable postoperative course. Otherwise, CSB was recommended for low-risk patients who can suffer a relatively comprehensive perioperative course, or who cannot be followed up for a long time since the events barely happened in the late stage of follow up.The key strengths of our study were that TBAD was the only indication for TEVAR and the characters of both groups were similar. However, the limitation of our study was the small sample, and the different follow-up time between groups , so the long-term result could be different as the follow up continued. Besides, due to non-randomization and a retrospective design, the study is subject to inherent bias and confounding. Further controlled studies are necessary to identify the first choice of LSA revascularization strategy for TBAD patients.


Lobectomy is the standard treatment for patients with stage I non-small cell lung cancer (NSCLC). Recent studies have shown promising results of stereotactic body radiation therapy (SBRT) in these patients. We retrospectively compared the outcomes of lobectomy and SBRT in these patients from our therapeutic center.Patients who underwent lobectomy or SBRT for clinical T1–2a (T size≤5 cm), N0 M0, NSCLC between December 2011 and August 2016 were reviewed. Patient characteristics, treatment-related outcomes and toxicities were analyzed. Propensity score matching (PSM) was performed to improve comparability between the two groups.n = 246) and SBRT (n = 70) group was 31.4 months and 24.9 months, respectively. Three-year local recurrence-free survival (LRFS) was comparable in the two groups . Recurrence-free survival (RFS) at 3-year in the lobectomy and SBRT groups was 85.4 and 69.5%, respectively (P = 0.014). Three-year overall survival (OS) after lobectomy and SBRT was 88.2 and 79.7%, respectively (P = 0.027), while 3-year cancer-specific survival (CSS) was 91.3 and 82.5% (P = 0.022). After PSM (45 matched patients in each group), there was no significant between-group difference with respect to 3-year LRFS , RFS , OS  or CSS . In matched subgroup, 30-day mortality after lobectomy was 2.2%, and no treatment-related death occurred after SBRT.Median follow-up period in the lobectomy (Treatment-related outcomes of SBRT and lobectomy were comparable. SBRT was well tolerated and had a very low toxicity profile in our study. SBRT is a promising alternative treatment option for stage I NSCLC patients. This study indicates that matching these disparate cohorts of patients is challenging. Clinical trials are essential to define the indications and relative efficacy of lobectomy and SBRT in a selected population. Due to the continuing growth of the geriatric population and the application of computed tomography (CT) screening, more patients with stage I non-small cell lung cancer (NSCLC) were diagnosed and underwent therapy in recent years . LobectoOver the past decade, the use of conventionally fractionated radiotherapy has increasingly been replaced by stereotactic body radiation therapy (SBRT) for patients who are not willing or unable to undergo surgery. Results of retrospective and phase II prospective studies have shown that local recurrence-free survival (LRFS) achieved with SBRT is approximately 90%, which is similar to that achieved with surgery in patients with operable stage I NSCLC –5. BesidWith increasing use of SBRT, the treatment model and patterns of care for patients with stage I NSCLC have witnessed a rapid change. Whether outcomes of SBRT in patients with stage I NSCLC are equivalent to those of surgery will continue to be debated in the absence of robust data from randomized trials. In geriatric patients and those with other risks for surgery, there is a need to evaluate the choice of treatment in Stage I NSCLC. In this study, we retrospectively compared the outcomes of SBRT with those of lobectomy in a cohort of patients with stage I NSCLC in our therapeutic center.Patients with clinical stage I NSCLC who were treated with lobectomy or SBRT between December 2011 and August 2016 at the Zhejiang Cancer Hospital were eligible for inclusion. The study protocol was approved by the medical ethics committee at the Zhejiang Cancer Hospital (No. IRB-2018-134). The inclusion criteria were: pathologically or clinically diagnosed NSCLC; confirmed as clinical Stage I (T1–2aN0M0) according to the TNM classification ; all patients accepted SBRT or lobectomy. Patients for whom histopathology results were not available were clinically confirmed as NSCLC based on the malignant growth characteristics of the primary lesion with an increase in the longest axis on repeat CT examination and hyper-metabolic activity on fluorodeoxyglucose-positron emission tomography (FDG-PET)  % and FEV1), and T stage. Matching was performed in a blinded manner  without replacement using a semi-automated method in the MatchIt package (version 4.8.3.4) for R (version 3.0.1) [Categorical data were compared using Fisher exact test; continuous data were compared using n = 246) or SBRT (n = 70). The baseline characteristics of the patients are shown in Table A total of 316 patients with clinical stage I NSCLC were included. These patients were treated with lobectomy (P = 0.133). Before treatment, 51 (72.9%) SBRT patients and 48(19.5%) lobectomy patients had histological confirmation. Forty-five (64.3%) patients in the SBRT group and 32 (13%) patients in the lobectomy group underwent FDG-PET scan.The median age of patients who underwent lobectomy was 67  years while the median age of those who underwent SBRT was 72.5  years. Patients with SBRT showed significantly poorer PS and lower FEV1. The proportion of clinical stage T1 in the SBRT group was similar to the lobectomy group , while the remaining patients underwent thoracotomy . Radiologically negative nodal involvement was detected as positive in 15 patients. Of these, 10 patients were upstaged to pathological N1, and 5 patients had N2 after surgery respectively, because of hilar and mediastinal lymph metastasis that was identified after lymph node sampling. Among these, 3 surgical patients received epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), and one patient received adjuvant chemotherapy only; none of the patients received adjuvant radiotherapy. In addition, 20 patients were upstaged to pathologic T2 tumors. None of the patients developed a synchronous tumor in a separate lobe at the time of surgery.In the SBRT cohort, different fractionation schemes Table  were useP = 0.768). Any new or growing lesions were closely assessed with repeat CT scan, in order to distinguish LR from fibrosis. LR was confirmed by histological examination (1 patient) or a combination of CT and FDG-PET (4 patients). Nine (3.7%) patients in the lobectomy group and 7 (10%) patients in the SBRT group developed RR during follow-up; the corresponding 3-year regional recurrence-free survival (RRFS) was 94.6 and 77%, respectively. RRFS was significantly better after lobectomy as compared to that after SBRT (P = 0.003)   months and 24.9  months, respectively. A total of 32 (13%) patients in the lobectomy group and 14 (20%) in the SBRT group developed disease recurrence within the observation time. Among these, 4 (1.6%) patients developed LR after lobectomy while 1 (1.4%) patient developed LR after SBRT; actuarial LRFS at 3 years was 97 and 91.7%, respectively    patients in the lobectomy group and 11 (15.7%) patients in the SBRT group died during the follow-up period. The survival outcomes in the lobectomy group were better than those in the SBRT group. The 3-year OS after lobectomy and SBRT was 88.2 and 79.7%, respectively (No treatment-related death was encountered in the SBRT group. Adverse events associated with SBRT included grade 1–2 pneumonitis in 27 38.6%) patients, and grade 3 pneumonitis in 1 patient. The other mild complications in 29 (41.4%) patients are detailed in Table 8.6% patiPSM resulted in a final cohort of 90 patients (45 lobectomy and 45 SBRT patients) who were eligible for further analysis. Patient characteristics are summarized in Table P = 0.635)     as compared to 89.6% (3 patients experienced LR) in the lobectomy group (P = 0.915). CSS was also similar between the two groups   pulmonary adverse effects, which did not affect everyday life.Our intent was to assess the difference between lobectomy and SBRT in stages I of NSCLC. In the past decade, SBRT has become the preferred treatment in the medically inoperable population due to high rates of local control, convenience, and suggestion of better survival compared to historic outcomes of conventional radiation. For low-risk patients with stage I NSCLC, lobectomy with mediastinal lymph node sampling/dissection is considered the standard care. However, for patients who are medically operable  but haveP = 0.44) [In this retrospective study, LRFS in the lobectomy and SBRT groups were both similar before and after PSM. In the past few decade, several retrospective and prospective studies had shown that SBRT achieved similar excellent LRFS in inoperable and operable stage I NSCLC , 10, 11. = 0.44) , which wThe present study effectively used PSM to balance the covariates in the two groups, which minimized the influence of confounding variables. Before PSM, patients in the lobectomy group achieved significantly better RRFS, DMRFS, RFS, OS and CSS than SBRT. The reason might be that patients who received SBRT were older and had significantly worse PS and pulmonary function. After PSM, no statistically significant differences were identified with respect to DMRFS, RFS, OS or CSS.In our study population, the main patterns of failure were RR and DM. There was significant between-group difference with respect to RR, while there was no significant difference with respect to DM or RFS after PSM. Firstly, this result suggests that SBRT patients may have been understaged before treatment due to the lack of invasive mediastinal and hilar staging and the lower proportion of patients who underwent FDG-PET scan in the study. Without these modes of diagnosis, the occult site is not easily detected with current imaging modalities. So PET-CT scans should be routinely performed prior to the treatment. Secondly, lobectomy with systemic mediastinal lymph node sampling/dissection showed an advantage over SBRT in terms of reduced incidence of RR. More importantly, the incidence of DM was lower in SBRT patients, which suggests that SBRT may offer benefit by targeting occult metastases, thereby leading to improved prognosis. To explain this, a study indicated the hypothesis that SBRT has unique mechanisms for improving immune function, which is mainly mediated by CD8+ T cells . So, a nP = 0.037) [P = 0.10); however, no significant difference was observed with respect to CSS with hazard ratios of 1.35 (95% CI: 0.70–2.62). However, Shirvani et al retrospectively analyzed 10,923 elderly patients from the SEER database with PSM, and found that the OS and CSS were similar in the two groups [In our study, there was no difference between SBRT and lobectomy with respect to 3-year OS and CSS. However, previous studies have yielded mixed results in this respect. In a recent Phase 2 clinical trial at the MD Anderson Cancer Center with long-term follow-up, the 7-year OS after SBRT was 47.5% . Notably= 0.037) . In spit= 0.037) . After Po groups . Others o groups . Therefoo groups . POSTLIVo groups . VALOR io groups .With respect to treatment-related toxicity, the published data and our findings suggest that SBRT is associated with a low incidence of mortality and morbidity compared with lobectomy . In prevThis study is limited by the short follow-up period of SBRT patients, which affected their survival rates. Because of this, OS may have favored SBRT. Longer follow-up is essential for adequate comparison of these groups and is ongoing. Another limitation is that fewer patients experienced pretreatment surgical staging in the SBRT group. The clinical staging of patients with NSCLC guidelines was often demonstrated as a poor predictive value by previous reports . To imprAfter matching with PSM for age, T stage, and other factors, the outcomes of LRFS, RFS, OS and CSS were not different between the two groups. SBRT offers a low-risk effective alternative for high-risk patients. However, treatment of these very high-risk patients with stage I NSCLC remains a challenge. Outcomes from findings in both treatment cohorts are strongly influenced by comorbidity and risk. Only randomized comparisons between lobectomy and SBRT will avoid the imbalances and selection biases present in this and other nonrandomized comparisons.


Acetate HD and HDF were superior when concerning IS elimination when compared to citrate HD and HDF. Moreover, residual diuresis was determined as the only predictor of lower indoxyl sulfate concentration, suggesting that it should be preserved as long as possible. This trial is registered with EU PAS Register of Studies EUPAS23714.Indoxyl sulfate has been identified as a major factor in the dysregulation of several genes. It is classified as a poorly dialyzable uremic toxin and thus a leading cause in the poor survival rate of dialysis patients. A monocentric, prospective, open cohort study was performed in 43 male patients undergoing chronic renal replacement therapy in a single hemodialysis center. The aim of the study was to determine the influence of acetate- versus citrate-buffered dialysis fluids in hemodialysis (HD) and postdilution hemodiafiltration (HDF) settings on the elimination of indoxyl sulfate. Also, additional factors potentially influencing the serum concentration of indoxyl sulfate were evaluated. For this purpose, the predialysis and postdialysis concentration ratio of indoxyl sulfate and total protein was determined. The difference was of 1.15 , 0.89 , 0.32 , and 0.44   Indoxyl sulfate (IS) is an important uremic toxin; therefore, its levels in plasma have been extensively studied in end-stage renal disease . Originain vitro [Indoxyl sulfate is a small solute MW = 213 g/mol) that is in >90% reversibly bound to plasma proteins, predominantly to albumin ; thus, t3 g/mol tin vitro . MoreoveIn this regard, increased dialysis time has not been effective . Furthervan der Waals forces; therefore, its dissociation constant is dependent upon ionic strength (μ), dilution, and pH [IS binding to albumin is driven by electrostatic and , and pH , 14. Tra, and pH , 16. Bec, and pH .μmol/l) is able to shift the albumin equilibrium towards a B conformation [in vivo is too complex to be extrapolated from in vitro experiments and a clinical study is therefore necessary.Moreover, citrate anions bind to albumin  and its ormation . This coormation . Thus, tThe aim of this study was to determine the influence of acetate- versus citrate-buffered dialysis fluids in hemodialysis (HD) and postdilution hemodiafiltration (HDF) settings on IS elimination considering, in addition, other factors potentially influencing the serum concentration of IS.A monocentric, prospective, open cohort study was performed in 43 male patients from a single hemodialysis center undergoing chronic RRT. All patients involved were under a maintenance dialysis regime for at least three months. The study focused primarily on assessing the serum concentration of IS, predialysis and postdialysis, and its relation to the dialysate-buffering component (acetate versus citrate) in two different modalities of hemodialysis—low-flux hemodialysis and high-flux hemodiafiltration. The RRT procedures used in this study are named ACET-HD, CIT-HD, ACET-HDF, and CIT-HDF, respectively.EUPAS23714.All procedures were approved by the Ethics Committee (Ref. number 2015 06 S25P) according to the Declaration of Helsinki (June 1964 and later amended). The included patients were properly informed of the study's aim and methods in both verbal and written form, and an informed consent was obtained for every dialysis protocol relevant to this study. The inclusion criteria encompassed maintenance dialysis for at least three months, willing and capable to cooperate with the procedures, clinical stability for at least a month before the start of the study, intradialytic hemodynamic stability, and outpatient basis of the treatment. The exclusion criteria included acute inflammation, malfunction of vascular access for dialysis, significant comorbidity such as cardiac failure, severe hepatopathy, and extrarenal complications of diabetes mellitus. This trial is registered with EU PAS Register of Studies Two commercially available dialysis solutions were compared in this study . The onl2) were used in both procedures, keeping a constant dialysate temperature of 36.0°C.Two RRT modalities were tested: conventional low-flux hemodialysis (HD) and online high-flux postdilution hemodiafiltration (HDF). Polysulfone dialyzer membranes , always as a first procedure in four consecutive weeks. The total study time was of 22 days, and the sequence of procedures was kept constant.A randomized sequence was considered unnecessary because midweek dialysis sessions were different between individuals, that is, 10 patients were treated using ACET-HD, 4 using CIT-HD, 27 using ACET-HDF, and 2 using CIT-HDF procedures.The study protocol also involved the collection of a blood sample  immediately before RTT and prior to heparin administration, using a standardized “slow-flow” method at the end of RTT in accordance with KDOQI guidelines . All samRoutine clinical chemistry parameters  were determined using an automated analytical system cobas 8000 . Citrate anions were determined using capillary electrophoresis PrinCE  and indirect detection by spectrometry at 375 nm using sodium chromate (10 mmol/l) in tetradecyltrimethylammonium bromide buffer (0.46 mmol/l).g for ten minutes, and serum aliquots were kept at −75°C until batch analysis.A simple analytical procedure was developed to determine IS potassium salt in blood serum by an ion-pairing ultrahigh performance liquid chromatography using an electrochemical detector . Blood samples were drawn into tubes containing a gel separator, allowed to clot for 30 min, centrifuged at 2000μl) were diluted with 350 μl ultrapure water and vortex mixed with 700 μl 7% perchloric acid to deproteinize samples and to release all bound IS into its free form [g/4°C. The supernatant was transferred to the glass autosampler vial and 10 μl injected into the liquid chromatograph.Serum samples (350 ree form . After 5μm) analytical column equipped with a guard column at a flow rate of 0.3 ml/min. The mobile phase was 85% phosphate buffer  and 15% methanol. IS was eluted at 5.5 minutes in an isocratic run. The detector voltage was set at +400 mV . The data is presented as median (interquartile range) or mean ± standard deviation. The statistical difference between the groups was tested using a Mann–Whitney rank-sum test, and n = 3), diabetic nephropathy (n = 18), glomerulonephritis (n = 11), interstitial nephritis (n = 9), kidney carcinoma (n = 1), and vasculitis (n = 1). The median duration of the dialysis treatment was of 4.0 years . The mean ideal body weight was of 82.5 ± 17.1 kg, body mass index of 27.9 ± 4.88 kg/m2 and body surface area of 1.99 ± 0.2 m2. Anuria was present in 48.8% of the patients, whereas the remaining patients had residual diuresis of 1010 ± 410 ml/day.Forty-three male patients aged 67.6 ± 11.5 years were included in the study. The underlying renal diseases found were polycystic kidney disease , and the equilibrated Kt/V index was 1.19 . The Kt/V index was not statistically different among the evaluated RRT procedures. The treatment data  are shown in The predialysis concentration of IS was similar before each RRT procedure; however, its distribution was quite scattered within the study group. Regardless, the predialysis concentration of 24 patients (55.8%) remained in the same tertile throughout the study, whereas 39 patients (90.7%) were found within the same tertile in 3 out of 4 conducted evaluations, reflecting steady dietary habits and stable large bowel microbiome .Further, residual diuresis was found as a positive influencing factor on the concentration of IS Figures  and 2; tWhen evaluating IS concentration during an RRT session, the acetate buffer hemodialysis modality was found to be superior to citrate buffer hemodialysis in decreasing its concentration (expressed as %) . Concernμmol/in CIT-HD and 336  μmol/in CIT-HDF versus 120  μmol/in ACET-HD and ACET-HDF (p = 0.03).Citrate serum concentrations after RRT were of 287  Prior to the first experimental procedure, total protein in serum was of 68.3  g/l, albumin 40.2  g/l, sodium 139  mmol/l, potassium 5.3  mmol/l, magnesium 0.9  mmol/l, total calcium 2.17 , and phosphate 1.74 . No correlation between IS levels and these parameters was found before the first experimental procedure was administered, probably due to the inherent large scatter of IS concentration in RRT patients.The present study, although small in scale, evaluated the influence of these buffering components over IS elimination; to the best of our knowledge, such reports are yet unavailable elsewhere. The most frequently used RRT modalities, hemodialysis and hemodiafiltration, were used during our tests while trying not to disturb the patient's RRT steady state during their normal schedule. Due to the ultrafiltration during HD and HDF procedures, a significant constriction in the distribution pool could be observed. Thus, IS concentration to total protein ratio was calculated and used in the statistical evaluations.When comparing HD and HDF in this study, the dialysis fluid containing acetate performed slightly better in hemodialysis without convection, contrary to previous trials which found negligible or only a slight improvement in hemodiafiltration. Anuric patients showed a significant improvement with the HD procedure, probably due to higher IS concentrations. This superiority was not observed in the residual diuresis group. In contrast to acetate, the fluid containing citrate was more efficient in hemodiafiltration, although only when expressed as a decrement (%) of the IS/total protein ratio. As mentioned in Introduction, “single session” studies are less sensitive to small changes in uremic toxin elimination, but long-term studies cannot distinguish between direct elimination and secondary metabolic effects of the RRT modality employed.The primary goal of this study was to compare both dialysates. Surprisingly, both RRT modalities performed much worse when using a citrate dialysate compared to that containing acetate. Slightly higher ionic strength was probably outweighed by the changes in albumin conformation caused by citrate anions and reduction of the free, dialyzable fraction of IS in either the dialyzer cartridge or the intravascular pool. A previously described slight pH and base excess increment during citrate HD or HDF may further increase the fraction of B conformer albumin and intensify the effect of citrate anions. In this regard, the significant influence of minor buffering components in the dialysis fluids on IS clearance is observed in this study for the first time.However, there was no difference in the Kt/V index according to the Daugirdas II equation, regardless of the cumulative blood flow intentionally kept constant in all procedures; therefore, the difference in IS elimination cannot be explained by the efficacy of the evaluated procedures. Further, several parameters were tested concerning their correlation with IS concentration; however, residual diuresis was the only significant factor found. Residual tubular secretion of IS results in its lower concentration in serum, which is beneficial for the patient; therefore, care should be taken in preserving residual diuresis as long as possible.Protein-bound toxins have been identified as an important problem in recent years; therefore, it is necessary to determine their concentration by means of an inexpensive procedure, allowing us to determine the elimination efficacy of protein-bound solutes by RRT and to identify those patients with excessive toxin levels. Thus far, up-to-date dietary protein restriction is the most effective intervention, balancing protein intake to prevent malnutrition and to eliminate the excess of aromatic amino acids.The results of this study draw attention to the influence of minor buffering components in dialysis solutions, providing an important and improved alternative to eliminate protein-bound toxins. Moreover, these results could direct the focus of research at the anionic components of dialysis solutions. However, this study was limited by its small-scale, single-session evaluation design and evaluation of only two types of dialyzer membranes. Also, the behavior of other protein-bound uremic toxins could be different and therefore needs to be assessed as well.The elimination of protein-bound toxins by contemporary RRT techniques remains inadequate. The elevated concentrations involved are associated with higher oxidative stress, endothelial dysfunction, and increased risk of cardiovascular events. Acetate-buffered dialysis fluids performed significantly better in HD as well as HDF procedures when compared with citrate-buffered fluids. Further, residual diuresis is a major factor in eliminating IS. These changes in buffering components are quite interesting to say the least, although they are yet an underestimated possibility in improving the inadequate indoxyl sulfate elimination during renal replacement therapy.