Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA.Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106–1); (ii) diluted template DNA from pure oocysts (×10–×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105–1 and 5 × 104–50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10–×1000).We assessed four types of DNA template used to generate standard curves in drug screening studies involving 6 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations.Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 10Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.For many studies involving  Cryptosporidium are a significant cause of enteric disease in humans and animals globally [Cryptosporidium spp. is ranked the fifth food-borne parasite in importance in Europe after Echinococcus spp. (E. granulosus and E. multilocularis), Toxoplasma gondii, and Trichinella spiralis based on the FAO/WHO criteria [Cryptosporidium spp. are transmitted via the faecal-oral route and oocysts may remain viable in the environment for long periods of time [®, Romark L.C., Tampa, USA) in humans and halofuginone lactate , and paromomycin  in calves, respectively. However, none of the drugs are able to completely prevent or cure the disease [Protozoan parasites of the genus globally . In humaglobally . Cryptoscriteria . Cryptos of time . Oocysts of time , 6. At p disease , 7.Cryptosporidium oocysts in stool or water samples for diagnostic purposes and water quality evaluations, to assess their infectivity, and to screen potential compounds for anti-Cryptosporidium efficacy . The. TheCryp studies , 11. Celed e.g. , 13). Th. ThCrypted  represents an important improvement over conventional PCR  with thiCryptosporidium qPCR quantification, vary from serial dilutions of template DNA extracted from a known stock concentration of oocysts [Methods employed to make controls that are used as templates to establish standard curves for  oocysts –23, seri oocysts , 25, tem oocysts –28, the  oocysts , or seri oocysts , 29.Cryptosporidium parvum is a proliferative parasite, undergoing both asexual and sexual division with infective sporozoites developing into trophozoites, meronts and 6–8 merozoites each (and therefore contain approximately 6–8 nuclei each) [Cryptosporidium oocysts. Such disinfection/inactivation studies utilising cell culture have previously been reported [ei each) , each ofei each) , 31. Thuei each) , 25. Simreported , 26, 28.relative, rather than absolute quantification, is relevant for compound screening and disinfection studies as only significant effects on parasites are relevant, the use of such standards could still be a feasible alternative. High variation is inherent within Cryptosporidium cell culture assays [Some studies utilise serial dilutions of free oocysts as standards for parasite samples after being grown in cell culture, or for parasite development in cell-free culture –24. As te assays  and coulIn qPCR reactions, the quantity of a specific DNA target in a sample is calculated from the observed quantification cycle (Cq) value and compared to the quantification cycle of known standards. It is important that the sample DNA is run in parallel under identical conditions with the group of controls. The accuracy of the data extrapolated from these experiments is therefore highly dependent on both the accuracy of the DNA standard quantification, and the quality of the standard curve generated Cryptosporidium spp. in vitro, we determined the differences in parasite DNA content using qPCR and data from Cq-values from four different standards: (i) serially diluted Cryptosporidium parvum oocysts; (ii) diluted template DNA from pure oocysts; (iii) equivalent concentrations of oocysts incubated in cell culture; and (iv) diluted DNA template from cell culture incubated parasites.Recently, digital droplet PCR (ddPCR) has been a method that somewhat overcame the variety of errors introduced when making these standard curves . In ddPCCryptosporidium parvum oocysts (Iowa strain) were purchased from Bunch Grass Farm  and stored in 50 ml phosphate-buffered saline (PBS) with penicillin (1000 IU) and streptomycin (1000 μg) at 2 × 107/ml (stock solution). According to the manufacturer, the oocysts were shed 14 days prior to delivery to the institute. The oocyst viability was assessed to be 82% via 4ʹ,6-diamidino-2-phenylindole/propidium iodide (DAPI/PI) stained oocysts in wet mounts immediately prior to inoculation of the first assay plate [ay plate , 34. Ooc2 at 37 °C.Human colon adenocarcinoma (HCT-8) cells (European collection of authenticated cell cultures (ECACC), Salisbury, UK) were cultured in maintenance medium  supplemented with horse serum (5% v/v), foetal bovine serum (FBS) (5% v/v), 1 mM sodium pyruvate and an antibiotic/antimycotic solution (penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (0.25 μg/ml)) . Cell sub-cultivation was performed twice per week with trypsin/ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich). Cells were incubated in 5% CO® 96-well plates (Sigma-Aldrich) (2 × 105 cells/well) and incubated in maintenance medium for 24 h until they reached confluence. Prior to infection of the cell monolayer, the oocysts were pre-treated as described by Slifko et al. [6 oocysts) was suspended in 1 part bleach solution, and 8 parts MilliQ water  for 10 min. The suspension was subsequently centrifuged at 4000×g for 4 min at 4 °C and the supernatant aspirated. The oocysts were then washed in 14 ml MilliQ water and centrifuged again , aspirated down to 200 μl and re-suspended in 1.8 ml maintenance medium pre-warmed to 37 °C to achieve a concentration of 106 oocysts/ml. The oocyst suspension was vortexed (10 s) and 100 μl  was subsequently diluted 10-fold to achieve 6 suspensions . Of each suspension, 100 μl was inoculated onto the cell monolayers (×5 repeats of each suspension) with a multi-channel pipette and incubated for 4 h . Maintenance medium was added to 5 blank wells with cell monolayers to control for potential oocyst contamination and inactivated oocysts in five replicates (5 × 105)  as a control for the washing steps. After incubation the oocysts suspensions and maintenance media were aspirated and washed once for 5 min (37 °C PBS) and 100 μl fresh maintenance medium  was added to the monolayers. Maintenance medium  was added to 2 wells containing no cells as a further control for contamination. Plates were subsequently incubated for 44 h. Oocyst inoculations were performed twice, on two separate plates, A and B. HCT-8 cells were on their 4th passage prior to use in the assays.HCT-8 cells were seeded onto Nunclono et al. . Briefly7/ml) were centrifuged at 4000×g for 5 min and the supernatant aspirated down to 200 μl to yield an oocyst suspension of 2 × 107/200 μl. This suspension was vortexed (10 s) and diluted 10-fold (MilliQ water) ×6 to achieve 7 suspensions: 106,105,104, 103, 102, 101 and 1 oocysts/10 μl in 1.5 ml Eppendorf tubes. This was performed twice, yielding two separate serial dilutions of oocyst suspensions. For each dilution, a 1.5 ml Eppendorf tube containing 200 μl of MillQ was prepared in parallel as a negative control.Stock oocysts . Subsequently, optical density (OD) was read at 450 nm. Cells were then washed 3 × 5 min  prior to cell lysis. Proteinase K (20 μl) and animal tissue lysis (ATL) buffer (180 μl)  were added to each well and incubated (56 °C) for 90 min. All well contents were aspirated into 1.5 ml Eppendorf tubes. For pure oocyst suspensions, Proteinase K and ATL buffer were added directly into 1.5 ml Eppendorf tubes containing 10 μl oocyst suspension. DNA was extracted using QiaCube  with the tissues and rodent tails protocol (elution volume 200 μl). DNA template from 106 of the pure oocyst suspension was then diluted 10-fold  with dH2O .After incubation for 44 h, the maintenance medium was aspirated and a water-soluble tetrazolium (WST)-1 assay  performed to assess cell viability as per manufacturer’s instructions. Briefly, 100 μl 10% v/v WST-1 solution in maintenance medium was added to each well and plates incubated for 30 min  and 0.5 μl DNA template. Two technical repeats were run for each sample in all assays. Six assays were run on the BIORAD CFX real-time instrument  using 96-well clear bottomed qPCR plates (BioRad). Six different qPCR runs were prepared: (i) and (ii) oocyst monolayer inoculations (cell monolayer plate A on qPCR Plate (i), and cell monolayer plate B on qPCR Plate (ii); (iii) stock oocyst serial dilution A and B; (iv) oocyst stock dilution A and B with their respective DNA template dilutions; and (v) and (vi) 3 biological repeats from the monolayer inoculations at the four highest inoculation concentrations , oocyst stock dilutions at the 5 highest suspension concentrations  and their respective DNA template dilutions. It was subsequently determined that differences between cell culture incubated oocysts and equivalent DNA template dilutions of the highest concentration would be valuable. DNA template from a separate oocyst titration study  were run on Plate (vii) alongside 5 × 104 ten-fold diluted template   in maintenance medium (100 μl) was seeded onto a 96-well plate with a multi-channel pipette without cells and inter-well variation assessed by flow cytometry with the NovoCyte NovoSampler , sample volume 60 μl, threshold set to Forward scatter -H (FS-H) < 300. Output was analysed with NovoExpress software and samples were gated based on blank control background readings to only include oocyst counts.A 10-fold dilution of oocysts (5 × 104–500) and equivalent concentrations of freely suspended oocysts were added to 190 µl Qubit™ 1× dsDNA HS Working Solution. Samples were vortexed (5 s) and centrifuged (5 s) prior to being analysed on an Invitrogen Qubit 4 fluorometer (Thermo Fisher Scientific). DNA content was recorded as ng/µl calibrated with Qubit™ 1× dsDNA HS Standard #1 and Qubit™ 1× dsDNA HS Standard #2 included in the kit.Qubit™ DNA quantification was performed with Qubit™ 1× double stranded DNA (dsDNA) HS Assay Kit . Ten microlitres of DNA template from each biological repeat from cell inoculation plate C . For plate B, 3/5 wells exhibited OD450 values > 75% (82.4 ± 6.5%), a threshold previously considered not to constitute toxicity in pharmaceutical screenings [450 values 66.9 and 9.1% when compared to the blank controls. Laboratory error resulted in marked pipette damage on the cell monolayer to the latter and was thus excluded from all subsequent analyses.A substantial loss in cell viability was observed in all wells inoculated with 105 to 1/well, respectively (R2 = 0.899). Two technical repeats  were clear outliers due to experimental error and were removed from the analysis. For Plate (ii) Cq values were: 28.91 ± 0.85, 30.43 ± 0.88, 34.73 ± 0.74 and 37.39 ± 0.6 for oocyst inoculates 105 to 102 (R2 = 0.901). Average Cq-values for one oocyst across biological repeats was 43.27 ± 1.26. For inoculates of 10 oocysts, amplification of DNA was observed in only one well for both technical repeats, Cq of 41.06 ± 0.77 for Plate (ii). Inactivated oocysts inoculated at 105/well indicated Cq-values across biological repeats of 36.34 ± 0.56 for Plate (i) and 37.39 ± 0.72 for Plate (ii). Cq-values for inactivated oocysts were not significantly different from those obtained from 100 stock oocyst inoculate (P = 0.7 and 0.99 for Plate (i) and (ii), respectively) but were significantly different for all other concentrations  were 28.76 ± 0.81, 29.69 ± 0.38, 33.45 ± 1.09, 36.5 ± 0.66, 41.28 ± 2.54 and 42.66 ± 1.19 for oocyst inoculates of 106, 105 and 104 oocysts, respectively for serial dilution A (R2 = 0.971). For 103 oocysts, one technical repeat failed to amplify any DNA with a Cq-value of 38 for the other. For serial dilution B, Cq-values were 28.74 ± 0.42, 30.29 ± 0.16, 33.86 ± 0.22 and 37.84 ± 0.75 for 106, 105, 104 and 103 oocysts, respectively. No signal was detected for DNA from 102, 101 and 1 for either dilution series (R2 = 0.968). No signal was observed for MilliQ water negative controls, as expected  and 27.62 ± 0.67, 30.35 ± 0.15, 33.64 ± 0.24, 37.95 ± 0.56 and 40.82 ± 0.34 for 106, 105, 104, 103 and 102 in serial dilution B (R2 = 0.956). Similar reductions in Cq-values were observed for corresponding dilutions of these templates: 31.04 ± 0.35, 34.09 ± 0.53, 38.11  and 40.82 ± 0.34 for ×10, ×102, ×103 and ×104 dilutions, dilution A (R2 = 0.987). DNA template dilutions of serial dilution B gave Cq-values of 30.52 ± 0.17, 35.11 ± 0.04, 38.19 ± 0.08 and 41.37  for ×10, ×102, ×103 and ×104 dilutions (R2 = 0.951). No DNA was amplified at the ×105 or ×106 dilutions. No signal was observed for the MilliQ water negative control . With Cq-values from all biological and technical repeats pooled, change in Cq (ΔCq) was 1.03 , 4.33  and 4.91  , 2.93 , 2.96   but not the pure oocyst suspension (P = 0.188). For 104 suspensions and the diluted template DNA equivalent, cell cultured oocysts had significantly lower Cq-values than both the stock dilution and template DNA dilution . For 103 oocysts, no difference was found between cell cultured oocysts and pure oocyst dilutions (P = 0.132) and no signal was observed in the equivalent diluted template. No signal was observed for 102 stock oocysts nor the respective diluted and the three technical repeats failed to amplify in the cell culture wells. No significant differences were observed between stock oocyst dilutions and the templates at the highest two template concentrations .Average Cq-values across biological repeats were 30.76 ± 0.5, 31.79 ± 0.48 and 36.18 ± 0.85 for 105–102 oocyst inoculates. With Cq-values from all biological and technical repeats pooled, ΔCq-values were 1.44 , 4.5  and 2 . The average ΔCq was 2.65 ± 1.33 , 2.85 , 3.7  and 4.71 . The average ΔCq was 2.9 ± 0.62 . ΔCq-values for the template DNA dilutions were 3.45 and 2.94 . The average ΔCq was 3.37 ± 0.31 ; for 104, both stock dilution and equivalent template dilutions having significantly higher Cq-values than cell culture  and for 103 . One hundred oocysts in cell culture produced significantly lower Cq-values compared to stock oocyst dilutions (P = 0.003) and the equivalent template dilutions (P = 0.001). No significant differences were found between the stock oocyst dilutions and their equivalent DNA template dilution with the exception of 104, where the Cq-value of the template was significantly higher than that of the stock dilution (P =. 0.0013). Inactivated oocyst Cq-values were not significantly different to values obtained from 102 oocyst inoculate in cell culture (P = 0.464).For Plate (vi), the average Cq-values across biological repeats in cell cultured oocysts were 29.21 ± 0.50, 30.66 ± 0.30, 35.16 ± 0.90 and 37.15 ± 0.86 for 104–50 oocyst inoculates  were 32.93 ± 0.68, 36.15 ± 1.07 and 39.89 ± 0.36 for ×10–×1000 cell cultured DNA template dilutions, respectively   but at lower seeding densities variation increased substantially .Flow cytometric analysis revealed relatively low variation for oocyst inoculates seeded at high concentration suspensions  in cell culture, compared to the diluted templates, but not for pure oocyst suspensions. For Plate (vi), all subsequent lower concentrations produced significantly lower Cq-values in cell culture compared to the stock oocyst and template dilutions, but for Plate (v), this was only the case for 104 oocysts. No significant difference in Cq-values between stock oocyst suspensions and cell culture were documented at 103 oocysts. The lack of significantly higher quantities of DNA in the cell culture samples at 105 oocysts may be attributable to the loss of cell viability in these wells although this is less likely to be a factor for Plate (vi). Cell stress as a result of a high oocyst inoculates is the likely cause of this loss of viability and a prospective study should assess any potential differences at lower oocyst inoculates. No DNA was amplified in any plates for stock oocyst dilutions and equivalent templates when there were < 100 oocysts.In plates (v) and (vi), cell culture Cq-values in many samples were significantly lower than values observed for stock oocyst dilutions and the equivalent template dilutions although this was not found uniformly across all oocyst concentrations. For 10The higher number of DNA copies obtained in cell culture inoculations compared with equivalent pure oocyst suspensions (or diluted DNA template of pure oocysts) is indicative of parasite growth. Once sporozoites have invaded the host cell, they undergo asexual and sexual multiplication and within 24 hours have produced meronts I  each con4 dilution despite these exact same templates amplifying DNA in Plate (iv). One hundred oocysts in cell culture on Plate (v) did not yield any Cq signal in one technical repeat of each well despite amplification being observed for the same templates in Plate (i) and Plate (ii), although one technical repeat for Plate (ii) failed to amplify for the ×104 template dilution in Plate (ii). Laboratory error during pipetting the template onto the qPCR plate may explain the lack of detection in instances where signal is not produced in one technical repeat for each well . This could include insufficient or no template being placed in the qPCR plate well. In order to mitigate this in the future it would be optimal to increase the quantity of DNA template in the reaction. As it was imperative that the same templates were used multiple times to insure robust comparison between methods, we were only able to use 0.5 µl in the present study. This may also explain the significantly higher Cq-value in the 104 equivalent template dilution on Plate (vi) when all other concentrations were not statistically different. Although Cq-values varied between plates, overall, there was a consensus between the same templates analysed on different plates.For plates (v) and (vi) the standard deviation (SD) between technical repeats was < 0.5 for all samples indicating high reproducibility . HoweverFor subsequent studies, variation in seeding densities of oocysts and excessive variation between Cq-values of technical repeats, automated techniques should be considered. These have the potential to reduce variation due to experimental error caused by human operators. However, these are expensive and therefore unavailable in many laboratories. If automated techniques are unavailable, studies should consider increasing the number of repeats, both biological and technical, in order to mitigate the problems caused by high variation.5–1) were not detected in the qPCR assay, indicating the complete removal of unexcysted oocysts and oocyst debris from the monolayer. Washing steps were similar with 3× PBS washes per well in both studies although Keegan et al. [5 inactivated oocysts were added to each well, this represents 99.9% oocyst inactivation and incubation for 10 min at 80 °C [Mean Cq-values for inactivated oocysts were not significantly different from that of the 100 oocyst inoculates in cell culture in all four independent plates in which they were included, i.e. (i), (ii), (iii) and (iv), with all other concentrations exhibiting significantly higher or lower Cq-values. This differs from the results obtained by Keegan et al.  where sen et al.  used 24-at 80 °C  may be p6 stock oocyst template did not yield significantly different Cq-values compared to stock oocyst dilutions except for the lower Cq-values in 104 stock oocysts and its equivalent diluted template in Plate (vi). Detection limits of stock dilutions of suspended oocysts were higher than previously reported with similar qPCR designs yielding sensitivity down to one oocyst or lower [C. parvum oocysts at numbers < 104. Furthermore, as oocyst numbers in a sample decreased, the percentage of positive signals produced for a particular quantity of oocysts also reduced for certain primers [Dilution of the 10or lower , 43. In  primers . It is iC. parvum cell culture [Cryptosporidium growth.Linearity values were also closer to 1 for both the stock oocyst dilutions and their equivalent templates than they were for cell culture derived templates. These linearity differences between the three DNA templates were maintained across all plates, further supporting the reproducibility of the assay. This is likely a consequence of the inherent variation encountered in  culture , which i5 and 104 oocysts compared to 104 and the subsequent dilutions (103–102) in plates (i), (ii), (v) and (vi). The reduced viability of the cells inoculated with 105 oocysts may in part be responsible for this finding; however, this was also observed in Plate (iv), where cell viability values of the 105 samples analysed all had OD450 values within 75% of the blank control wells. This could suggest that possible inhibitors are present in the reaction mix; however, it is unlikely that the inhibitors originate from the host cells as these were seeded at the same density for all samples. The reduced ΔCq at the highest concentrations of stock oocysts and diluted template DNA was not as marked as in cell culture, although was still lower than differences between lower concentrations. This was initially thought to be a result of human DNA in the cell culture standards exceeding the binding capacity of the DNA extraction column resulting in < 100% C. parvum DNA present in the extracted template. However, DNA content in cell culture standards from plate C did not exceed 10.64 ± 0.56 ng/µl . The DNeasy® Blood & Tissue Kit (Qiagen) is capable of extracting, on average, 30 µg DNA from lysed 25 µg tissue sample [6 stock oocysts producing significantly lower Cq-values than 105 oocysts in cell culture.As shown in Figs. 6 oocysts and if inhibitors were present, lower Cq-values would be expected in the diluted template [Cq-values from serially diluted oocysts did not differ significantly from the DNA template dilutions of 10template . Howevertemplate , so it sC. parvum does not appear to multiply in a linear fashion in cell culture [4 oocyst cell culture standards on Plate (vii) yielded significantly less DNA at ×10 and ×1000 dilutions  than oocysts incubated in cell culture  will make it more difficult to interpret relative parasite inhibition based on the standard curve. Some studies have utilised oocyst suspensions as standards for  culture , 23. As  culture , 41.C. parvum previously and these have been compared to in vivo models [5 would be appropriate for cell culture assays. However, this should be considered in light of the propensity of increased variation and ΔCq in cell culture at lower oocyst inoculates. As only relative growth inhibition is required for many assays involving C. parvum it would appear the pure oocyst suspensions or diluted DNA templates may represent ideal standards at present. Since cell culture derived standards will more accurately reflect actual DNA content from parasite grown in cell culture however, there is a clear incentive for prospective study to focus on the mechanisms explaining this reduced linearity.To facilitate inter-laboratory comparisons of qPCR results, standardised protocols are required including procedures for generating standard DNA templates if precise data are needed. Although numerous studies have reported DNA content from cell cultured o models , in the Additional file 1: Table S1. Cq-values of quantitative PCR Plate (i). Cryptosporidium parvum oocysts inoculated onto HCT-8 cell monolayer plate A. Two technical repeats for each well . Abbreviations: TR, technical repeat; BR, biological repeat; SD, standard deviation.Additional file 2: Table S2. Cq-values of quantitative PCR Plate (ii). Cryptosporidium parvum oocysts inoculated onto HCT-8 cell monolayer plate B. Two technical repeats for each well . Abbreviations: TR, technical repeat; BR, biological repeat; SD, standard deviation.Additional file 3: Table S3. Cq-values of quantitative PCR Plate (iii). Pure C. parvum oocyst serial dilution A and B. Two technical repeats for each Eppendorf . Abbreviations: TR, technical repeat; SD, standard deviation.Additional file 4: Table S4. Cq-values of quantitative PCR Plate (iv). Pure C. parvum oocyst serial dilution A and B and equivalent template dilution A and B. Two technical repeats for each Eppendorf . Abbreviations: TR, technical repeat; SD, standard deviation.


Haplotype analysis showed that CTLA4 Crs733618Crs16840252 might increase the risk of GO . In conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial.Graves’ disease (GD) is an autoimmune inflammatory disease, and Graves’ ophthalmopathy (GO) occurs in 25–50% of patients with GD. Several susceptible genes were identified to be associated with GO in some genetic analysis studies, including the immune regulatory gene CTLA4. We aimed to find out the correlation of CTLA4 gene polymorphism and GO. A total of 42 participants were enrolled in this study, consisting of 22 patients with GO and 20 healthy controls. Chi-square or Fisher’s exact test were used to appraise the association between Graves’ ophthalmopathy and CTLA4 single nucleotide polymorphisms (SNPs). All regions of CTLA4 including promoter, exon and 3’UTR were investigated. There was no nucleotide substitution in exon 2 and exon 3 of CTLA4 region, and the allele frequencies of CTLA4 polymorphisms had no significant difference between patients with GO and controls. However, the genotype frequency of “TT” genotype in rs733618 significantly differed between patients with GO and healthy controls (OR = 0.421, 95%CI: 0.290–0.611,  Graves’ disease (GD) is an autoimmune inflammatory disease. The annual incidence of Graves’ disease is 20 to 50 cases per 100,000 persons  but has Genetic analysis has identified several susceptible genes, including genes encoding thyroglobulin, thyrotropin receptor, HLA-DRβ-Arg74, protein tyrosine phosphatase nonreceptor type 22 (PTPN22), cytotoxic T-lymphocyte–associated antigen 4 (CTLA4), CD25, and CD40 . Recent Treatment of GO varies according to the disease severity . EstabliTwenty-two patients with GO  and 20 healthy controls  were included in Linkou Chang Gung Memorial Hospital during the period from June 2017 to May 2019.® DNA Mini kit  according to the manufacturer’s instructions. DNA concentration and purity were evaluated by measuring the optical density at 260 and 280 nm through UV spectrometer.The blood samples were collected in EDTA-coated vacuum tubes. The genomic DNA was extracted using QIAamp2O. The PCR mixture contained 1 µL DNA, 10µL HotStarTaq DNA Polymerase , 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM) and 12 µL ddHThe PCR production was purified by mixture containing 2.5 µL shrimp alkaline phosphatase and 0.05 µL exonuclease I , and the purified PCR products were sequenced using ABI PRISM 3730 DNA Analyzer . Single nucleotide polymorphisms (SNPs) analysis were performed on CTLA4 gene, and rs11571315, rs733618, rs4553808, rs11571316, rs62182595, rs16840252, rs5742909, rs231775, and rs3087243 were selected for genotyping.All statistical data analyses were performed by the SPSS 17.0. Genotype and allele frequencies of CTLA4 gene for patients with GO were compared to healthy controls. Chi-square or Fisher’s exact test were used to evaluate the association between GO and CTLA4 SNPs.Among 22 GO patients, there were 14 females and 8 males, and the average age of patients was 46 years old. Moreover, half of GO patients was moderate in severity categories , and theThe severity of GO was classified via the criterion proposed by Werner . Class 0p > 0.05) (p = 0.043), and the “CC” and “CT” genotype in rs16840252 were nearly significantly differed in genotype frequency (p = 0.052). Haplotype analysis (p = 0.043).All SNPs were in accordance with the Hardy-Weinberg equilibrium in the control group ( > 0.05) . There w > 0.05)  of “TT” analysis  showed tp = 0.052. As a result, the two-marker combination was found to be more significantly related to GO (p = 0.025).Our data suggested that the genotype of −1722 (rs733618) in the promoter region of CTLA4 was associated with GO, the p value of TT vs. (CT + CC) was 0.043. We further combined −1722 (rs733618) and −1147 (rs16840252) together to observe the correlation between GO and controls, because the genotype of −1147 was very close to the critical point, Previous research had found that rs733618 , rs23177p = 0.012) [W. Tang et al., used expectation-maximization (EM) algorithm to create several feasible haplotypes, and haplotype compounded of Grs733618Crs16840252Ars231775Grs3087243 was associated with gastric cardia adenocarcinoma (= 0.012) . Our res= 0.012) . The rs1= 0.012) . Investi= 0.012) ,33,34 an= 0.012) ,36 were Several studies in GD-related CTLA4 SNPs were summarized in Herein, Crs733618Crs16840252 of the CTLA4 promoter was shown to be associated with increasing risk of GO. Promoter polymorphism can influence gene transcription, as well as the disease severity . The sigIn conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial.


Moraxella catarrhalis OMVs may orchestrate the susceptibility of pathogenic bacteria and yeasts to cationic peptides (polymyxin B) and serum complement. Using growth kinetic curve and time-kill assay we found that OMVs protect Candida albicans against polymyxin B-dependent fungicidal action in combination with fluconazole. We showed that OMVs preserve the virulent filamentous phenotype of yeasts in the presence of both antifungal drugs. We demonstrated that bacteria including Haemophilus influenza, Acinetobacter baumannii, and Pseudomonas aeruginosa coincubated with OMVs are protected against membrane targeting agents. The high susceptibility of OMV-associated bacteria to polymyxin B excluded the direct way of protection, suggesting rather the fusion mechanisms. High-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV) and zeta-potential measurement revealed a high sequestration capacity (up to 95%) of OMVs against model cationic peptide accompanied by an increase in surface electrical charge. We presented the first experimental evidence that bacterial OMVs by sequestering of cationic peptides may protect pathogenic yeast against combined action of antifungal drugs. Our findings identify OMVs as important inter-kingdom players.The virulence of bacterial outer membrane vesicles (OMVs) contributes to innate microbial defense. Limited data report their role in interspecies reactions. There are no data about the relevance of OMVs in bacterial-yeast communication. We hypothesized that model  Pseudomonas aeruginosa or Myxococcus xanthus [M. catarrhalis OMVs carrying β-lactamases confer protection to M. catarrhalis and Streptococcus pneumoniae against β-lactam antibiotics [E.coli can protect β-lactam-susceptible E.coli and fully rescue them from β-lactam antibiotic-induced growth inhibition [P. aeruginosa it was reported that variation in physicochemical properties was dependent on growth phase , influencing, therefore, the cell association activity [Moraxella catarrhalis Mc6 [Candida albicans against antifungals drugs. Finally, we investigated whether OMVs-dependent protection is a signature of only free vesicles or those associated with the cell.In the environment, different microbial populations, including bacteria and fungi, co-exist. Mixed microbial populations are also a common feature in many diseases. Hence, understanding which factors may be potentially important players in interspecies dynamics of growth and to what degree these dynamics are mediated by the host is very important. Among these factors are outer membrane vesicles (OMVs) of Gram-negative bacteria classified recently as secretion system type zero . These p xanthus ,13,14. Oibiotics . Likewishibition . Severalhibition ,8,17. Nehibition  but alsohibition ,20. The activity . All afoalis Mc6  as modelM. catarrhalis 6 had 30–200 nm (Moraxella immunoglobulin D-binding protein), CopB, MhuA (hemoglobin-binding protein), TbpA (transferrin-binding protein A), TbpB (transferrin-binding protein B), LbpB (lactoferrin-binding protein B), OMP M35, and MipA .As shown in transmission electron microscopy (TEM) image, the diameters of outer membrane vesicles from 0–200 nm a. The re5–106 cfu/mL of prominent human pathogens, including nontypeable Haemophilus influenzae, Pseudomonas aeruginosa, and Acinetobacter baumannii, was in the range 0.5–5 μg/mL, and caused killing effect within 2 h (Polymyxin B (PB) was used as a model of cationic peptide. In our study, using 4-h time-kill assay, the minimal bactericidal concentrations (MBC) of PB against 5 × 10thin 2 h a. When pH. influenzae appeared to be serum-sensitive. Two others, A. baumannii and P. aeruginosa, were serum-resistant. The resistant strains showed either 100% survival or only a slight decrease (within one order) after 4 h of incubation in 50% or 75% NHS. Nontypeable H. influenzae strains were sensitive to NHS-dependent killing with a reduction of cfu/mL in the range of six orders after 30 min of incubation (NTHi3) or 120 min of incubation (NTHi6) in the presence of 25% or 50% serum, respectively, compared to the initial inoculum. These strains co-incubated with NHS and 20 μg/mL of OMVs exhibited a time-dependent increase in survival, which was similar to growth of control incubated in the presence of heat-inactivated serum (HiNHS).To define the influence of OMVs on the survival of cross-pathogens in the presence of active serum (NHS), the 4 h complement bactericidal assays were performed b. Of theA. baumannii incubated only in the presence of active NHS was slightly killed by the lytic complement action within 4 h, whereas after parallel exposure to OMVs, its growth was at the level of HiNHS control. P. aeruginosa was highly resistant to 75% action of serum, thus co-incubation with OMVs did not change the growth dynamics (data not shown). When control bacteria were incubated in the presence of heat-inactivated NHS, neither killing nor other growth alterations were observed for any of the tested strains, indicating that observed lysis was complement-dependent. Overall, these findings indicate that OMVs efficiently protect serum-sensitive gram-negative pathogens against complement action while accelerating the growth of moderately serum-resistant strains.Candida albicans to combined action of both drugs. Our results showed, that for ~105 cfu/mL of this yeast, the MIC50 for fluconazole (FLC) and MIC100 for polymyxin B (PB) were, respectively, 1 µg/mL and 128 µg/mL. Next, based on checkerboard assay, we assessed the growth of C. albicans incubated with polymyxin B alone, fluconazole alone, or their combinations at various concentrations. The results revealed that polymyxin B at concentrations much lower than MIC (1/8 MIC and 1/16 MIC) exerts a potent antifungal effect against C. albicans when combined with 1 µg/mL (MIC50) of FLC  of FLC a.1-PB8 and FLC1-PB16 (μg/mL) are fungicidal, which was expressed by one order and by two orders of decrease in viability, respectively  showed almost complete inhibitory activity against the formation of hyphae. FLC0.0625-PB4 had also clear inhibitory effect on yeast-to-hyphae transition in comparison to FLC alone. The presence of 20 μg/mL OMVs in all tested drug combinations caused a significant increase in the percentage of hyphae-forming cells , as a result of membrane permeabilization, we showed that OMV-dependent protection was dose-dependent and that PB was very quickly depleted from the environment in the presence of at least 20 µg/mL OMVs leading to a decrease in peptide activity  and over 96% (p < 0.001), respectively, in reference to standard PB . Similar to PB, the strong permeabilizing potency of membrane attack complex (MAC), present in 10% human serum, against E.coli ML35p was considerably decreased or even dumped as the concentration of OMVs increased  to make sure that the entire cell population could be covered . We founociation a,b. Thesl assays c,d, as wl assays e,f were M. catarrhalis associated and not associated with OMVs, the lethal effect of PB occurred after 4 h  formation away from target bacteria and thus protect them from MAC-mediated lysis. The intensity of OMV-dependent complement activation in vitro was correlated with the number of free vesicles in the environment. Therefore, it is possible that any fluctuation in the number of released vesicles may positively or negatively influence complement activation of this potent innate mechanism.M. catarrhalis species while they did not work in interspecies systems. By evidencing the lethal effect of cell-associated OMVs exposed to polymyxin B, we showed that OMVs do not protect against AMPs this way, suggesting a fusion between OMVs and OM rather than only shielding. It is tempting to speculate that the specificity of interaction between OMVs and recipient cell is somehow pivotal in its sensitization at least to AMPs. We are therefore in agreement with Tashiro et al. that elucidating the selectivity in OMV-cell interaction is critical for an improved understanding of the outcome of this reaction. On the other hand, it has been documented that interactions with OMVs for some cross-pathogens can be specific, less specific, or not specific at all [Next, using the PB-dependent model, we investigated whether OMVs-dependent protection is a signature of only free vesicles or those associated with the cell. Using flow cytometry, we documented that the association of OMVs with bacterial cells may be very potent and species-specific, influencing the cell sensitivity to antimicrobial compound. Our vesicles were able to associate only with representatives of c at all . Thus, tC. albicans. Polymyxins alone are effective against C. albicans only at relatively high concentrations [C. albicans in the presence of both drugs and OMVs using FLC at MIC50 (1 µg/mL) and PB in the range much lower than MIC (1/8–1/16). To our knowledge, for the first time, we have provided evidence that bacterial OMVs can inhibit the fungicidal action of certain combined antifungal agents that are effective against pathogenic yeasts. Next, we documented that sub-MIC concentrations of FLC (1/16) and PB (in the range of 1/8–1/32) applied together weakened or almost completely inhibited the yeast-to-hyphae transition. Furthermore, in the presence of OMVs, the filamentous phenotype was considerably recovered and even strengthened despite exposure to both drugs. It is a very undesirable action of OMVs, since filamentation increases the virulence of C. albicans, which in hyphae form is more invasive and can attach in a higher number to epithelial cells than yeast and pseudohyphae forms [C. albicans less vulnerable to destruction. The documented inter-kingdom OMV-based mutualistic relationship between bacteria and yeasts are, to our knowledge, a novel phenomenon. Therefore, its importance for more complex in vitro and in vivo conditions, as well as pathophysiology, remains to be clarified.Next, we address the question of whether OMV-dependent trapping of cationic antimicrobials can protect pathogens from other kingdoms such as fungi of the species trations . Azoles trations . They aftrations ,31. It htrations . Previoutrations ,33. Therae forms ,35. UsinCandida–bacteria interactions are not a rare occurrence and may have an important impact on the human disease by causing e.g., the faster biofilm growth or Candida-dependent induction of antimicrobial resistance of Staphylococci [P. aeruginosa or A. baumannii with C. albicans or other fungi may lead to the killing of yeast [C. albicans, which compromises the immune system, facilitates the emergence of A. baumannii pneumonia [Candida spp, could be an alternative for treatment [The complex ylococci ,37. Someof yeast ,39. It cof yeast . The synneumonia . The intreatment ,42. Likereatment ,44,45. Freatment ,47, it i3 cfu/mL. In our study, 20 μg/mL of OMVs was protective against 106 cfu/mL, indicating that OMV amounts used in our study are rational and may resemble the infectious condition.Furthermore, there is many data on how different stress factors may induce bacterial hypervesiculation incuding temperature stress [Another important issue of our results is the number of OMVs needed to show biological activity. In general, the information about the number of OMVs produced in vivo is still very limited. Although OMV production in the course of infection has been documented , the mage stress , oxidatie stress , hyperose stress , or antie stress . So far,e stress ,53,54 ane stress . Based oCandida virulence. This work may serve as an important basis for further evaluation of OMVs-dependent interactions within pathogenic bacterial-fungal communities. Our results indicate that OMVs are important players in interspecies and cross-kingdom microbial interactions.Overall, our results on the protective and somehow deleterious for pathogens role of OMVs, in the bacteria–bacteria and bacteria–yeast interspecies systems, underline the enormous potential of these nanostructures as accelerating factors in case of various mixed infections. Furthermore, the OMV-dependent mode of actions may serve as a model of passive resistance of gram-negative bacteria not only to antimicrobials, but also to humoral defense components, which operate to disrupt cell membrane. Likewise, for dimorphic yeasts, the ability of OMVs to sequester membrane active compounds that augment the antifungal activity of azoles may have an important impact on 2+, Mg2+ ; RPMI 1640 ; o-nitrophenyl-β-D-galactopyranoside , heat inactivated  FBS .). HPLC chemicals: Acetonitrile  for separation was HPLC far UV/gradient grade ; 32 mM Na2SO4 solution for chromatographic usage was prepared with 4.5 g anhydrous sodium sulfate  and MiliQ .BHI  ); TSB ; TSA , Bradford reagent ; β-nicotinoamide adenine dinucleotide hydrate ; fluconazole , hemin ; polymyxin B sulfate salt ; fluorescein isothiocyanate ; Hank’s Buffer with CaMoraxella catarrhalis , nontypeable Haemophilus influenzae , Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (PAO1), Candida albicans (Ca1), mutant of Escherichia coli ML-35p, a lactose permease-deficient strain with constitutive cytoplasmic β-galactosidase. All strains were from the collection of our Institute. M. catarrhalis strains were grown on Columbia agar plates or BHI broth. NTHi strains were grown on chocolate agar plates or in BHI broth supplemented with hemin and NAD at final concentrations of 15 μg/mL each. Moraxella and Haemophilus strains were cultivated at 37 °C with 5% CO2. A. baumannii; E. coli ML-35p and P. aeruginosa were routinely cultured in TSB medium at 37 °C. C. albicans was cultured in yeast extract-peptone-glucose (YPG) in 37 °C.The following microbial strains were used: Outer membrane vesicles (OMVs) isolation was performed as described previously . The pro5–106 cfu/mL) was incubated with or without PB (in the range 0.5–5 µg/mL) or NHS (in the range 25%–75%) with the presence or absence of free OMVs (2 µg/mL or 20 µg/mL) in the final volume of 200 µl 1% medium (w/v). The experiments were performed from 0 to 240 min and the 10 µl aliquots of 10-time diluted bacterial suspensions were plated in triplicate on appropriate agar plates at 0, 30, 120, and 240 min time points. The colony counts and cfu/mL were calculated the next day. The controls for NHS contained heat-inactivated serum (HiNHS), . The bactericidal activity of PB or NHS was expressed in each time point as cfu/mL in reference to cfu/mL in time 0. Analogous experiments with bacteria associated and non-associated with OMVs (20 µg/mL or 80 µg/mL) in the presence of PB (5 µg/mL) were performed. Analogous experiments for selected combinations of antifungals were carried out for C. albicans except that: (i) Initial inoculum was ~2 × 105–5 × 105 cfu/mL, (ii) tested antimicrobials were PB (8 µg/mL or 16 µg/mL) and fluconazole (1 µg/mL), (iii) the medium was 0.5% YPG (w/v), and (iv) incubation lasts 24 h. All microbicidal assays were performed at least two times in triplicate.For testing PB or human serum (NHS) activity, the log-phase bacterial suspension  in 1% BHI (w/v) and 60 min intervals for yeast at 105 cfu/mL  diluted in 0.5% YPG.All growth kinetics experiments were performed on the flat-bottomed 96-well microplates  at 37 °C in volume 200 µL. Dynamic of growth was measured using Varioskan™ LUX reader with measurements at 30 min intervals for bacteria at 108 cfu/mL exponentially growing bacteria were washed by centrifugation with HBSS Ca2+ Mg2+ and resuspended in 100 µl OMVs (20 µg/mL or 80 µg/mL) in Eppendorf tube. Association was performed for 30 min at 37 °C with gentle mixing. Thereafter, all samples were washed with HBSS Ca2+, Mg2 by centrifugation  to remove free OMVs particles, diluted to 2 × 106 cfu/mL with HBSS Ca2+, Mg2, and used in growth kinetics assay.OMV-associated bacteria preparation: 0.5 mL of ~10C. albicans was ~105 cfu/mL and cells were incubated at 37 °C without shaking. Synergy/growth potentiation was tested by the checkerboard method including a two-dimensional array of serial concentrations of both drugs. The fluconazole was used in concentrations 1–64 μg/mL whereas polymyxin B in concentrations 1–128 μg/mL. Wells without drugs or yeast inoculation were included as positive and negative controls, respectively. The MIC100 of polymyxin B and the MIC50 of fluconazole was defined as the lowest drug concentration that caused a decrease in absorbance of 100% and 50%, respectively, compared to control in drug-free medium.The microdilution assay was performed on flat-bottom microplate according to the CLSI (formerly NCCLS) standard  except tC. albicans cells were preincubated overnight in YPG supplemented with sub-MIC concentration of FLC at 37 °C without shaking. To induce hyphal transition, the suspensions of C. albicans (OD = 0.4) were treated with 10% heat inactivated FBS in PBS for 2 h at 37 °C in 24-well flat-bottom microplates (NUNC) in volume 0.4 mL, in the presence of sub-MIC concentrations of FLC (1/16) and PB (range 1/8–1/32) and with shaking (130 rpm). The samples were observed under inverted microscope Zeiss Axio Vert. A1 with objective Zeiss LD A-Plan (40 × /0.55 Ph1). The images wer recorded using Industrial Digital Camera 5.1 MP 1 /2.5”. The assessment of cell morphology and the length (μm) of hyphae was performed using ImageJ software.Before the induction of filamentation, M. catarrhalis OMVs in vitro, the time-dependent decrease of permeabilizing activity of this peptide against E. coli ML-35p with constitutive β-galactosidase activity was measured using the ONPG-mediated β-galactosidase microplate assay as previously described [6 cfu/mL) in 10 mM sodium phosphate NaPB (pH 7.4) were incubated in the flat-bottom 96-well microplates  with 5 µg/mL of PB in the presence of OMVs at concentration range 1–20 µg/mL and with 3 mM ONPG as β-galactosidase substrate in the final volume of 150 μL. Microplates were incubated at 37 °C for 1.5 h and optical densities at λ = 405 nm were measured every 15 min (spectrophotometer ASYS). All the assays were performed at least 3 times in duplicates.To assess the polymyxin B sequestration by escribed . The finM. catarrhalis OMVs in vitro, the various concentrations of OMVs (1–20 μg/mL) were pretreated with active normal human serum (NHS) at a volume ratio 1:9 and the soluble terminal complement complex SC5b-9 was measured using ELISA kit according to manufacturer’s instructions . Unstimulated NHS served as negative controls.This method was described previously . To asseg for 10 min at 4 °C to remove PBS. The collected OMVs were reconstituted with 500 μL of 0.05 M carbonate/bicarbonate buffer (pH 9.5) and washed by centrifugation on vivaspin as described before. The collected OMVs at concentration 80 µg/mL were labeled with 1 mg/mL FITC at carbonate/bicarbonate buffer for 30 min at 37 °C with gentle mixing in the dark. The remaining fluorochrome was rinsed of 3 times with a 500 µL of cold carbonate/biscarbonate buffer each time, using 50 kDa Vivaspin. The final FITC-labelled OMVs were resuspended in Hank’s buffer Ca2+, Mg2+ at original concentration.OMVs labeling: Initially, OMVs (80 µg/mL) in PBS were concentrated using 50 kDa Vivaspin centrifugal concentrators  at 14,000 × 550 = 0.2 was centrifuged and subsequently washed with 1 mL of HBSS Ca2+, Mg2+ . The pellet was resuspended in 200 µL of OMV-FITC conjugate (80 µg/mL OMVs) and incubated for 30 min at 37 °C with gentle mixing in the dark. Afterward, samples were washed with HBSS Ca2+Mg2 by centrifugation  to remove free OMVs particles and finally resuspended in 200 µL of HBSS Ca2+ Mg2.OMVs association with bacteria: 1 mL of each fresh bacterial culture corresponding to OD® easyCyte flow cytometer . Before analysis, the samples were diluted 1:100 to obtain approximately 1–5 × 105 cell/mL in HBSS Ca2+ Mg2+. Fluorescence intensity of bacterial cells associated with OMVs was analyzed for green fluorescence in the FL1 channel, by collecting 5000 events. Data were expressed as mean fluorescence intensity (MFI). Data analysis was performed using InCyte Merck Guava software .Flow cytometric analysis: To detect bacterial cells associated with FITC-labeled OMVs flow cytometry analysis was performed using GUAVAThe Zeta potential of OMVs was measured at room temperature (25 °C) by a Zetasizer Nano-ZS 90 . The instrument was equipped with a Helium–Neon laser (633 nm) as a source of light. The detection angle of Zetasizer at aqueous media was 173°. Considering the influence of factors such as conductivity  or pH of the solution on the Zeta potential, to minimize their influence, all Zeta potential measurements were performed with 10 mM NaPB buffer (pH 7.4) or in MiliQ.2SO4 in water (pH 3.2 achieved with H2SO4) in isocratic separation mode. Eluent flow 0.75 mL/min and detection realized with a UV detector at 215 nm. Column and sample temperature were respectively 30 °C and 5 °C; separation run time was 15 min. The injection volume was 50 µL. The calibration curve was in the range of 1.56 µg/mL to 100 µg/mL prepared with 10 mg/mL standard stock solution of polymyxin B sulfate salt. Each concentration was injected twice daily for precision into 3 or 2 independent samples, and between day eight samples. The linearity of the method was in a range from 6.25 to 100 µg/mL with 16% of the average coefficient of variation (CV%) for the sum of peaks, and with a coefficient of determination R2 = 0.997. Since the major constituents of polymyxin B are B1 and B2 [HPLC chromatography of polymyxin B was performed as described previously ,59. The To quantify the results, the relative concentration was calculated as the mean PB concentration of sample/mean PB of control × 100%. Results were expressed as mean ± SD.g for 15 min at RT to remove OMVs. The filtrate was collected and stored at −20 °C until use. Tested and control samples were processed identically.Sample preparation: 200 µL of reaction mixtures containing OMVs (5 µg/mL or 20 µg/mL) and PB (50 µg/mL) or PB alone (50 µg/mL) were incubated for 30 min at 37 °C with gentle mixing. The samples were ultrafiltrated using 50 kDa vivaspin centrifugal concentrators  at 14 000× −1) was added per 20 mg of OMV proteins and incubated in a heating block at 60°C for 1 h. The presence of LOS in proteinase K-treated OMV samples was analyzed by dodecylosulfate gel electrophoresis. The 15 µL of samples were applied to the Glycine-SDS-PAGE (10%) gel path, corresponding to 5 µg LOS. Electrophoresis was carried during 2.5 h at a constant voltage of 80 V at 4 °C. The gel was fixed for 1 h at room temperature using a fixing solution (EtOH: Acetic Acid: MiliQ: 40:5:55); POCH, Gliwice, Poland) The fixed gel was stored overnight at 4 °C. MiliQ was exchanged for fresh before visualization using a modified Tsai and Frasch [The concentration of lipooligosaccharide (LOS) from Mc6 OMVs was determined based on the purpald assay . Dilutedd Frasch  silver sw/v) aqueous solution of uranyl acetate. The OMVs were imaged with a TEM operating at an acceleration voltage of 150 kV .The OMVs preparation for TEM was described previously . OMVs wep < 0.05.The data were expressed as the mean ± SD, and analyzed for the significant difference by one-way ANOVA or Kruskal–Wallis ANOVA rang using the Statistica (version 13.1) software  Differences were considered statistically significant if


Prasiola japonica ethanol extract, we consider the former to be an important compound used in the cosmeceutical industry.Loliolide is a monoterpenoid hydroxylactone present in freshwater algae that has anti-inflammatory and antiaging activity; however, its effects on ultraviolet-damaged skin have yet to be elucidated. This study investigated the antiapoptosis and wound-healing effects of loliolide using HaCaT cells (a human keratinocyte cell line). Loliolide inhibited the expression of reactive oxygen species (ROS) induced by ultraviolet radiation as well as wrinkle formation-related matrix metalloproteinase genes and increased the expression of the damage repair-related gene SIRT1. The apoptosis signaling pathway was confirmed by Western blot analysis, which showed that loliolide was able to reduce the expression of caspases 3, 8, and 9, which are related to ROS-induced apoptosis. In addition, Western blotting, reverse-transcription polymerase chain reaction (PCR), and real-time PCR analyses showed that loliolide enhanced the expression of the epidermal growth factor receptor signaling pathway  and migration factors, such as K6, K16, and K17; keratinocyte growth factor; and inflammatory cytokines, such as interleukin (IL)-1, IL-17, and IL-22 expressed during the cellular scratching process, suggesting a putative wound-healing ability. Because of the antiapoptosis and antiscratching effects on skin of both loliolide and loliolide-rich  Skin is a self-renewing organ that defends the body against external threats such as heat, infections, mechanical or chemical insults, and ultraviolet (UV) radiation ,2. UV raSince the skin is the outer barrier of our body, another of its functions is to protect our body from potential injury and, when injury occurs, to promote quick restoration to prevent potential infections . TherefoWound healing begins with the induction of an inflammatory response due to bacterial colonization and barrier disruption, which stimulate the immediate production of interleukin (IL)-1, tumor necrosis factor alpha (TNF-α), and Th17 cell-produced cytokines  can impaPrasiola japonica ethanol extract (Pj–EE) is a loliolide-rich source that has also been shown to possess antioxidant activity [Loliolide  is an acactivity . Therefo2) condition -2,5-diphenyltetrazolium bromide (MTT) assay, HaCaT cell viability was not affected even up to a loliolide concentration of 100 µM under normal cell culture conditions a. By conondition b. Howeveondition b. Next, ondition c. In addondition d. To conondition e. Furtheondition f.p < 0.05 compared with the normal group and ** p < 0.01 compared with the control group.We examined the expression of oxidative stress-induced MMPs and Sirt-1 in HaCaT cells to determine loliolide’s involvement in senescence and cell death processes. Loliolide was found to reduce the expression of MMP-1 a, MMP-9 To determine the wound-healing effects of loliolide in HaCaT cells, cell migration was measured. In this respect, we confirmed that loliolide (100 µM) showed an increase in cell migration and wound scratch closure in HaCaT cells a,b. To dWe examined the antiscratching effects of Pj–EE, a freshwater green algae whose main compound is known to be loliolide, in keratinocytes (HaCaT cells). Firstly, cell viability in HaCaT cells was measured during Pj–EE-treated condition. According to MTT assay, the viability of the cells was not decreased by Pj–EE until concentrations of up to 400 µg/mL a. To meaAs the outmost layer of the human body, our skin is constantly exposed to UV radiation. UVB radiation 280–320 nm), one of the most damaging types of solar UV emissions, can affect various skin structures, causing wrinkling, roughness, and premature aging 0–320 nm,. ConstanIn addition to presenting a good antiapoptotic effect, loliolide also dose-dependently reduced not only oxidative stress d, but alWound healing is an essential physiological process that involves responses such as inflammation, blood clotting, cellular proliferation, and extracellular matrix (ECM) remodeling, with the final aim of preventing infection and dehydration in the body . The lon2DCF–DA and the Annexin V-FITC Apoptosis Detection Kit were purchased from Sigma-Aldrich Chemical Co. . TRIzol reagent was purchased from Thermo Fisher Scientific . The primer sets for the polymerase chain reaction were synthetized by Macrogen , while PCR premix was purchased from Bio-D Inc. . Primary antibodies to phospho-, total or cleaved forms of PI3K , AKT , ERK , caspase 3 , caspase 8 , caspase 9 , and β-actin (CST #4967), and horse radish peroxidase (HRP)-labeled secondary antibodies to antimouse and antirabbit IgG were obtained from Cell Signaling Technology .Loliolide (purity: 98% by HPLC) was purchased from Chemfaces . HaCaT cells, a spontaneously transformed aneuploidy immortal keratinocyte cell line from adult human skin, were purchased from the American Type Culture Collection . MTT, fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco . HPrasiola japonica used in the experiment was supplied by the Prasiola japonica Research Center in Samcheok City, Gangwon-do, Korea. Samples were cut into 2 × 2 cm2 pieces and then extracted with 70% ethanol at room temperature for 24 h. Samples and solvents were extracted at a gGa ratio of 1:20 (w/v). Following extraction, the filtrate was filtered through a 110-nm filter paper , and the filtrate was concentrated using a vacuum concentrator . The concentrated samples were dried using a vacuum freeze dryer  and the yield of the dried samples was measured. The final weight of the extract was 2.752 g , with a yield of 6.13%. The dried samples were stored in a −20 °C freezer until use. Analysis of loliolide in Pj–EE was performed by gas chromatography, as reported previously. (https://patents.google.com/patent/KR101591401B1/ko).HaCaT cells (a human keratinocyte cell line) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% humidified incubator.4 cells per well in a 96-well plate for 24 h and then treated with loliolide (0–100 µM) and Pj–EE (0–400 µg/mL) for 24 h. For testing UV-protective activity, HaCaT cells were pretreated with loliolide (0–100 µM) and then further exposed to UVB radiation (30 mJ/cm2). The viability of HaCaT cells treated with loliolide or Pj–EE or UV-irradiated HaCaT cells pretreated with loliolide was measured using the MTT assay, in which cells were first incubated with 10 µL/well of MTT solution  for three hours and then treated with 100 µL of MTT-stopping solution (10% sodium dodecyl sulfate with 10% HCl). After eight hours, the absorbance of the solubilized formazan was measured at 570 nm using an optical density reader . For determining the compound’s cytotoxicity, the same method was applied using only PJ–EE (0–100 µg/mL) for 24 h [HaCaT cells were seeded at a density of 4 × 10for 24 h ,33.5 cells/mL in a 12-well plate containing previously sterilized, round glass cover slips. After 24 h, cells were treated with loliolide (0–100 µM) for 30 min, washed with PBS, and exposed to UVB (30 mJ). To quantify ROS generation, cells were stained with H2DCF–DA  (10 µg/mL) for 30 min in dark conditions. Cells were washed with PBS twice, fixed with 1 mL of 3.7% paraformaldehyde in PBS for 10 min, and the cover slip was then mounted on a rectangular glass slide using mounting solution and left to dry at room temperature for 24 h. Samples were examined using a Nikon Eclipse Ti fluorescence microscope [HaCaT cells were seeded at a density of 4 × 10croscope   for 30 min, washed with PBS, and exposed to UVB (30 mJ). Cells were washed twice with PBS and fixed with 1 mL of 3.7% paraformaldehyde in PBS for 10 min. Cells were washed with PBS two more times, stained with DAPI reagent  (1 µL/mL) for 30 min, and then washed with PBS two more times. The cover slip was then mounted on a rectangular glass slide using mounting solution and left to dry at room temperature for 24 h [HaCaT cells were seeded at a density of 4 × 10for 24 h . Samples2). For staining, cells were washed twice with cold PBS and resuspended in 1× binding buffer at a concentration of 106 cells/mL. Afterwards, 100 μL of suspension (105 cells) was transferred to E-tubes, 10 μL of propidium iodide and 5 μL of fluorescein isothiocyanate–Annexin V  were added, and cells were incubated for 15 min at room temperature in the dark. Finally, 400 μL of 1× binding buffer  was added and fluorescence was assessed using a Guava easyCyte flow cytometer .Apoptosis was analyzed by flow cytometry after different cell treatments. Cells were pretreated with or without loliolide (0–100 µM) and then further irradiated with UVB (30 mJ/cm5 cells/mL) and cultured in a 5% CO2 incubator at 37 °C for 24 h. Cells were treated with loliolide (0–100 µM) or Pj–EE (0–200 µg/mL) and incubated for 24 h at 37 °C in a 5% CO2 incubator. The cells were then washed three times with cold PBS and lysed with lysis buffer . The lysates were centrifuged at 12,000 g for eight min, and the supernatant was transferred to another tube. Protein quantification was performed by Bradford analysis of the supernatant. Cell lysates (supernatant) quantified by Bradford analysis were analyzed by Western blot. Phosphorylation or the total levels of PI3K , p-PI3K , AKT , p-AKT , and β-actin , and cleaved or total levels of cleaved caspase 3 (CST #9664. 1:2000), procaspase 3 , cleaved caspase 8 , procaspase 8 , cleaved caspase 9 , and procaspase 9  were evaluated in HaCaT cells. All Western blot data in this study are representative of two experiments showing similar patterns.HaCaT cells were seeded in a six-well plate  or Pj–EE (0–200 µg/mL). The cells were then exposed by UVB (30 mJ/cm2) and further incubated for 24 h. TRIzol reagent was used to extract mRNA according to the manufacturer’s instructions [HaCaT cells were seeded at 6 × 10ructions . The con5 cells/mL) and cultured in a 5% at 37 °C in a CO2 incubator for 24 h. Scratch wounds were created mechanically with a sterile pipette tip (Ø  =  0.1 mm), whose size range varied from approximately 0.5 mm to 0.9 mm in width. Detached cells and debris were washed away with PBS solution, and fresh medium was treated to the wells. Cells were treated with loliolide (0–100 µM) or Pj–EE (0–200 µg/mL) and incubated for 24 h.HaCaT cells were seeded in a six-well plate .All data are presented as the mean ± standard deviation of at least three independent experiments. A Kruskal–Wallis/Mann–Whitney U test was used to compare statistical differences between the experimental and control groups. A


Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in  Escherichia coli. However, these findings have recently been challenged . This suggests that these markers actually respond to ATP decreases and that the rare stationary-phase-like cells in a growing population have low energy levels. These cells showed considerably higher antibiotic tolerance after being sorted out from a growing population, showing that they are persisters. Importantly, dropping ATP to stationary-phase levels with arsenate treatment in a growing culture recapitulates the persister level of a stationary-phase population, showing that low energy is sufficient for tolerance. If low ATP levels result in tolerance, then high ATP levels should have the opposite effect. Indeed, supplementation of tryptic soy broth (TSB) medium with glucose increased ATP levels significantly and resulted in a 100-fold reduction in persisters. We observed similar results linking ATP and persisters in a study of E. coli  cycle enzymes are tolerant of killing by antibiotics.Toxin-antitoxin (TA) modules and the stringent response have been proposed as mechanisms of antibiotic tolerance, based primarily on studies of allenged –4. Similormation . In sear E. coli . On the S. aureus exhausts glucose and expression of TCA cycle enzymes is upregulated  TCA cycle enzymes and in late, gudB (glutamate dehydrogenase), sucA (2-oxoglutarate dehydrogenase), sucC (succinyl-coenzyme A [CoA] synthetase), fumC (fumarate hydratase) TCA cycle enzymes by transducing insertions from the Nebraska Transposon Mutant Library into methicillin-susceptible S. aureus (MSSA) strain HG003, which is susceptible to antibiotics  and membrane potential . (G) Quantification of membrane potential. A total of 4,721 cells were subjected to membrane potential analysis. Download Copyright © 2019 Zalis et al.2019Zalis et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bacillus PS3 F0F1 ATP synthase. The sensor absorbs at 405 nm and 488 nm and emits at 513 nm. At higher levels of ATP, there is increased fluorescence under conditions of 405-nm excitation and decreased fluorescence under conditions of 488-nm excitation. A ratio between the two emission signals reports ATP concentration. This ratio does not depend on the amount of the reporter, eliminating errors due to variations in QUEEN levels among cells. We first cloned QUEEN in plasmid pEPSA5 under the control of a xylose promoter, but the fluorescence signal was weak. We then performed codon optimization of a QUEEN construct for expression in S. aureus, which yielded an improved fluorescence signal  cultured at 30°C in TSB without glucose . Samples were washed with PBS, placed on top of a PBS–1% agarose pad, and observed under the microscope. The two fluorescent signals corresponding to 405ex  and 488ex  were sequentially collected one after the other. Scale bar, 5 μm. Download FIG S2, DOCX file, 0.8 MB.Optimization of Copyright © 2019 Zalis et al.2019Zalis et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus and the gltA, gudB, sucA, sucC, and fumC mutants. The frequency distribution of the ratios is shown in gltA was similar to that of the wild type (Using fluorescence-activated cell sorter (FACS) analysis, we monitored ATP in single cells of wild-type ild type , while sild type . Howeverild type , explainPersisters tend to wake up during sorting, which makes sorting prior to antibiotic treatment problematic , 5. Sortuvrgfp in plasmid pALC1434  and ATP   or in TSB without glucose  or in Mueller-Hinton broth (MHB) as indicated. Cultures were grown at 37°C with shaking at 225 rpm. Strains encoding the QUEEN construct were grown on tryptic soy agar (TSA) plates with chloramphenicol 10 μg/ml at 30°C or in TSB without glucose with shaking at 225 rpm at 30°C with chloramphenicol 10 μg/ml to maintain the pEPSA5 plasmid. QUEEN expression was induced with 0.03% xylose. High concentrations of xylose led to a growth defect. MSSA strain HG003 was used for these studies, and the mutations for all TCA cycle genes in this background were transduced from the Nebraska Transposon Mutant Library (NTML) using bacteriophage 80α or φ11. Mutations were subsequently confirmed by amplification of the sequence from the beginning or end of the gene of interest to the transposon insertion as previously described , and ampicillin 100 μg/ml was used to maintain plasmids where necessary.g for 7 min at 4°C, washed twice in phosphate-buffered saline (PBS), and pelleted again. Pellets were resuspended in 500 μl PBS, transferred to a 2-ml tube, washed a final time, and then flash frozen with liquid nitrogen and stored at –80°C. Bacterial pellets were resuspended in 100 mM ammonium bicarbonate and lysed by vortex mixing 5 times for 1 min and then with 0.1-mm-diameter zirconia/silica beads with resting periods of 30 s on ice. Samples were then digested with trypsin as previously described (18 solid-phase-extraction (SPE) cartridges  . Peptide concentrations were measured by bicinchoninic acid (BCA) assay (Thermo Scientific).Four replicates were prepared from either exponential-phase or stationary-phase cells, and cells were grown for 4 or 24 h, respectively, after overnight cultures were diluted 1:1,000 in 100 ml MHB. Cells were grown in 500-ml baffled flasks at 37°C with shaking at 225 rpm. Cells were pelleted at 5,000 × escribed  and desam/z was used for the isolation of ions with a collision energy level of 28%. Tandem MS (MS/MS) spectra were recorded at a resolution of 17,500. The resulting data were processed using MaxQuant V1.5.2.8. . Matches between runs and MaxLFQ data were used for quantification; other parameters were conserved by default. Proteins identified only by site, reverse hits, and contaminants were removed. Only protein groups with a measured label-free-quantitation (LFQ) intensity level in at least 60% of the examples of one sample type were conserved for further quantification. LFQ intensities were log2 transformed and median normalized. Analyses of statistics  and normalization steps were performed in R using the Stat packages.For each sample, a 0.5-μg volume of peptides was separated using a 200-min gradient and a Waters nanoACQUITY ultraperformance liquid chromatography (UPLC) system  coupled with a Q Exactive HF mass spectrometer . MS scans were recorded at a resolution of 35,000. The top 12 ions from the survey scan were selected by the use of a quadrupole mass filter for high-energy collision dissociation, and mass data were analyzed by the use of an Orbitrap analyzer. A window of 2 1.5.2.8. . Protein−1, respectively). To enumerate survivors over time, 100 μl of culture was removed, pelleted by centrifugation, washed with 1% NaCl, serially diluted, and plated on TSA, and CFU counting was performed after 24 h of regrowth on TSA to enumerate survivors. Experiments were performed in biological triplicate.Overnight cultures were diluted 1:1,000 in 2 ml TSB  in a 14-ml capped culture tube  and grown 5 h to the late exponential phase, and starting CFU were plated. Details regarding the proteins identified and the enrichment can be found in Table S1 in the supplemental material. Cultures were challenged with ciprofloxacin, oxacillin, or gentamicin  pEPSA5- pEPSA5-QUEEN2m was created by amplifying QUEEN2m from pRsetB-his7-QUEEN2m (GAATTCTAGGGAGAGGTTTTAAACATGAAAACTGTGAAAGTGAATATAAC-3′) and Q2m-R (5′-CGAGCTGGTACCTCACTTCATTTCCGCAACGCTC-3′) and cloning it into the EcoRI/KpnI sites of pEPSA5 downstream of a xylose promoter (sarA gene has been added in Q2m-F primer (indicated here in bold).-QUEEN2m  with pripromoter . RestricQUEEN2m(opt).(ii) pEPSA5-QUEEN2m(opt) was codon optimized from the original sequence by using the JCat tool (QUEEN2m in a custom plasmid. A fragment containing QUEEN2m(opt) and its RBS was excised from this plasmid with EcoRI/KpnI and cloned into the EcoRI/KpnI sites of pEPSA5  were cultured at 30°C in TSB without glucose  to the stationary phase, inoculated into fresh medium of the same composition at 1:100, and cultured for 4.5 h at 30°C. Samples were washed with PBS, placed on top of a PBS–1% agarose pad, and observed under a Zeiss LSM 710 confocal microscope using a 63× oil immersion lens objective. The two fluorescent signals, 405ex and 488ex, were sequentially collected. Differential interference contrast (DIC) images were recorded alongside the 405ex acquisition. Images were acquired by the use of Zen software at a resolution of 1,024 by 1,024 pixels, with a lane average of 8, and were processed with Fiji software  or 26 h (gudB and sucC mutants) to reach comparable levels of CFU per milliliter. Xylose 0.03% was used to induce expression of QUEEN. The strains used was as follows: HG003 pEPSA5 (empty vector), HG003 pEPSA5-QUEEN2m, gltA::ϕΝΣ pEPSA5-QUEEN2m, gudB::ϕΝΣ pEPSA5-QUEEN2m, sucA::ϕΝΣ pEPSA5-QUEEN2m, sucC::ϕΝΣ pEPSA5-QUEEN2m, and fumC:: ϕΝΣ pEPSA5-QUEEN2m. Three biological replicates were analyzed for each strain.Flow cytometry was carried out using an Attune flow cytometer. Spatially separated violet and blue lasers were used for excitation at 405 nm and 488 nm, respectively, to calculate the ratio of the level produced at each of these excitation wavelengths to that seen with emission at 513 nm. To prepare samples, –80°C stocks were grown on TSA plates with chloramphenicol 10 μg/ml at 30°C. Single colonies were selected, and overnight cultures were inoculated into TSB without glucose plus chloramphenicol 10 μg/ml with shaking at 225 rpm at 30°C. Tubes were inoculated 24 h later from overnight cultures and grown for 25 h  and side scatter (SSC) and then on the basis of GFP fluorescence (GFP-A). Gates were set to include the brightest 5% and dimmest 5%, in addition to a middle level (∼30% of the population). Cells were sorted from each population onto TSA plates. Plates were incubated at 37°C for 24 h, and colonies were counted. Recorded colonies were normalized for expected colony counts by the use of an untreated control sort plate. Percent survival was calculated from the dim, middle, and bright GFP fractions.Cell sorting was carried out using a BD FACSAria II flow cytometer with a 70-μm-inner-diameter nozzle. Briefly, 1:1,000 dilutions of overnight cultures were grown 5 h to late exponential phase and challenged with ciprofloxacin (10× MIC) for 24 h. After 24 h, cells were diluted 1:100 and sonicated as previously described  to dispeBacLight bacterial membrane potential kit according to the manufacturer’s instructions. Briefly, cultures were grown 5 h in TSB medium and diluted to 106 CFU/ml in 1 ml filtered PBS. DiOC2(3)  was used to stain cells for 30 min before samples were analyzed by flow cytometry. The ratio of mCherry fluorescence to GFP fluorescence was calculated for each cell. The negative-control sample was pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min before staining was performed. For flow cytometry analysis, cells were gated by size, and then ratios were calculated.Membrane potential was measured using a PXD013151 and on MassIVE under the identifier MSV000083600 (ftp://massive.ucsd.edu/MSV000083600/).The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the data set identifier


This 6-month follow-up study aims to determine 7-day personal ambient NO2 and BTEX exposure levels via compact passive diffusion samplers in female participants from Cape Town, and investigate whether exposure levels are associated with cardiovascular risk markers. Overall, the measured air pollutant exposure levels were lower compared to international standards. NO2 was positively associated with systolic and diastolic blood pressure (SBP and DBP), and inversely associated with the central retinal venular equivalent (CRVE) and mean baseline brachial artery diameter. o-xylene was associated with DBP and benzene was strongly associated with carotid intima media thickness (cIMT). Our findings showed that personal air pollution exposure, even at relatively low levels, was associated with several markers of cardiovascular risk in women residing in the Cape Town region. Exposure to ambient NO Ambient air pollution is a global health concern and is associated with numerous adverse health effects including cardiovascular disease (CVD) ,2,3. The2 and PM with a diameter of ≤2.5 µm (PM2.5) as the air pollutants that are most dangerous to public health [The World Health Organization (WHO) has identified gaseous pollutants such as NOc health . Other gc health ,16,17,18c health ,16,17,18c health . 2 and BTEX exposure during two 1-week time periods in a repeated-measurements study (6-month follow-up) of apparently healthy women residing in the Cape Town region of South Africa, and (2) determine whether current levels of personal NO2 and BTEX exposure are associated with markers of cardiovascular risk, including blood pressure (systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively), flow-mediated vasodilatation (FMD), retinal blood vessel widths, and carotid intima media thickness (cIMT). Reports on air pollution levels and related health effects are mostly based on data from the developed world, while the situation in developing countries remains under-reported ,21,22,23The current study formed part of a larger parent study called EndoAfrica and participants for the current study were recruited from the healthy control study group of the parent study . Ethical2 device , a RadielloTM BTEX passive diffusion sampler , and an ACR SmartButton® temperature logger  placed in the external mesh pocket of a backpack , and the average temperature (°C) for the 7-day period was recorded. Once collected, the NO2 and BTEX samplers were sealed, stored at 4 °C and sent for quantification according to the manufacturer’s protocol. Each participant was equipped with a Gradko rapid NObackpack a–d. The backpack e. Althoubackpack f. Partic2 samplers were sent to Gradko International Ltd. laboratories (United Kingdom Accreditation Services (UKAS) accredited)) for quantification according to UKAS method GLM 7 [2−)). The color reagent was prepared as previously described [2 concentrations were determined via chemiluminescence ultraviolet (UV) spectrophotometry . Calibration standards and linearity checks were used to calibrate the spectrophotometer, and mid-range and zero standards were analyzed at intervals throughout the sequence for quality assurance. The calibration curve was used to calculate the NO2- concentration for each sample. The ambient NO2 concentration was calculated from NO2− concentrations and expressed as µg/m3 [NOod GLM 7 . A calibescribed  and usedas µg/m3 . TM glass tube containing the cartridge  and 12.5 µL 2-fluorotoluene internal standard ). The tube was stirred for 30 minutes on a rotational shaker. BTEX quantification was performed by means of gas chromatography (Thermo Trace) and a mass spectrometer (Thermo DSQ II with helium as carries gas (a constant flow of 1 mL/min)). A cross-bond diphenyl/dimethylpolysiloxane column  with a 1.4 µm-thick film was used for sample separation . Equipment calibration was performed by injecting the standard solution ) at 0.03 to 30 µg/g before analysis. Sample concentrations were calculated from chromatograms using a standard curve. The limit of detection (LOD) was calculated as 3.3 (standard deviation (SD) of areas/slope). Samples with conc. <LOD were not presented. Results were expressed as average NO2 and BTEX exposure concentration (μg/m3) for the 7-day measuring period. The same procedure was followed for the 6-month follow-up visits.BTEX samplers were sent to the Flemish Institute for Technology and Research  for quantification as previously described ,31. BTEXTM, Cipla DIBCARE, Bellville, South Africa) and collected fasting blood and mid-stream urine samples. Cardiovascular measurements included SBP and DBP  via an Omron M6 automatic digital blood pressure monitor . Hypertension was defined as either SBP of ≥140 mmHg or DBP of ≥90 mmHg, based on the South African Hypertension Society guidelines [2 height.At each visit, participants completed a comprehensive questionnaire to collect data on age, smoking status (defined as smoking or non-smoking), socioeconomic information , and lifestyle . Qualified research nurses measured fasting blood glucose levels (mmol/L) by finger prick . This specific chemiluminescence analysis was based on the highly sensitive Near Infrared Particle Immunoassay rate methodology where anti-CRP antibody-coated particles bind to the CRP in the serum sample resulting in an insoluble aggregate formation and turbidity. Samples were prepared for analysis by adding 4.5 µL serum sample, 42 µL antibody-coated particles (particle-bound goat and mouse anti-CRP antibody), 125 µL buffer 4 and 42 µL diluent (sodium azide: <0.1% (w/w)). The hsCRP concentration was determined automatically as the rate of aggregate formation . For the purposes of this study, increased hsCRP was defined as hsCRP levels of >3 mg/L based on previous reports suggesting that hsCRP levels above this cut-off value are associated with increased cardiovascular risk [Fasting whole blood was collected in blood collection tubes  Ltd.; Milnerton, Western Cape, SA). Serum was extracted from whole blood samples and stored at −80 °C. Serum samples were analyzed by the South African National Health Laboratory Service , a South African National Accreditation System (SANAS)-accredited laboratory, for high-sensitivity C-reactive protein (hsCRP) levels by means of an IMMAGElar risk . ®-c analyzer and CREP2 kit; Roche Diagnostics, Basel, Switzerland). The principal of the method was based on the enzymatic formation of hydrogen peroxide  that reacted with 2,4,6-triiodo-3-hydroxybenzoic acid for quinone imine chromogen. Creatinine levels were determined by the color intensity of the quinone imine chromogen .Mid-stream urine samples were obtained at each visit, placed on ice and immediately delivered to the NHLS for the quantification of urinary creatinine levels by means of chemiluminescence (cobasv:v)) MU-d4 and 3 , and 4 MHA-d7  with 465 µL 1% acetic acid . A matrix-matched calibration curve was applied for the quantification of HPMA, BMA, and PMA to compensate for the matrix effect. To achieve this, spiked urine samples were used containing 10 µL urine, 25 µL mixed internal standard ) and 445 µL 1% HAc in ultra-pure water. Additional mid-stream urine samples were stored at −80 °C and sent to VITO  for level determinations of the following urinary metabolites: N-acetyl-S-(3-hydroxypropyl)-L-cysteine , N-acet−)). An Acquity UPLC® high-strength silica T3 column  with UV detection (Photodiode array (PDA) detector set at 259 nm) was used for the simultaneous quantification of the urinary metabolites [v:v). Levels of metabolites were calculated based on the corresponding matrix-matched calibration curve.Twenty microliters of each sample was injected in an ultra-performance liquid chromatography /mass spectrometry  with an Esaote Doppler probe  connected to computerized software with edge detection technology  as previously described [Vascular endothelial function was assessed via FMD of the right brachial artery, 3–4 cm proximal to the elbow. FMD was measured in the supine position with a mobile Esaote MyLabescribed ,39. BrieTM Five portable ultrasound device  and a B-linear-mode Esaote Doppler probe  connected to computerized software  specific for the determination of carotid metrics. Measurements were taken 5 mm proximal to the dilation of the carotid bulb. The mean of the left and right carotid diameter and mean of the left and right cIMT were calculated and used for statistical analysis.The cIMTs of the left and right carotid arteries were determined by B-mode ultrasonography as previously described ,41. cIMThttps://mona.health) by trained investigators using a standardized protocol as previously described [Additionally, retinal images were captured with a Canon CR2 digital camera  and analyzed with semi-automated software . Depending on data distribution, a paired sample Student’s t-test (parametric) or Wilcoxon signed-rank test (non-parametric) was used to identify significant differences between baseline and follow-up visits. A Spearman’s Rho correlation (nonparametric data) was used to identify correlations between variables. To determine the association between NO2 or individual BTEX compounds or total BTEX  and different cardiovascular outcomes, a linear mixed model (LMM) regression analysis was used with participants nested in each visit. Variables with skewed data distribution  were log-transformed for regression analysis. To evaluate personal air pollution effects on cardiovascular outcomes independent of potential confounding effects, we selected a priori covariates that are known determinants for cardiovascular outcomes and variables with a potential link with personal air pollution exposure and cardiovascular outcomes. These include age, BMI, smoking, socio-economic status (reflected by employment), sleep, and ambient temperature [2 or BTEX or total BTEX as an exposure variable, participants at each time point as a random effects factor variable with random intercept to account for possible inter-individual variation while adjusting for age, BMI, date of assessment visit, and average temperature as continuous fixed effects and smoking status, employment status, and hours of sleep at night as fixed categorical variables. For effects on other vascular outcomes, we additionally adjusted LMMs for SBP. To determine estimated effects on % FMD and cIMT, the mean brachial diameter and carotid diameter were additionally adjusted for, respectively. Q-Q plots of the residuals were used to test the assumptions of linearity. The significance threshold was set at p < 0.05 for all statistical analysis.All statistical analyses were performed with IBMperature ,45,46,47A total number of 77 female participants were recruited for the study. Sixteen participants who completed their baseline visits did not consent to continue with the 6-month follow-up visit and were excluded from the study. A total number of 61 healthy female participants (mean ± SD age at baseline: 42.5 ± 13.4 years) of mixed ancestry completed both assessment visits . 2. Most participants  reported that they sleep 6 to 9 hours per night. The mean ± SD SBP and DBP values (SBP: 122.5 ± 19.9) mmHg, and DBP: 84.2 ± 12.0 mmHg) were within the normal range  exhibited elevated hsCRP levels. The majority of participants were current smokers , but none reported any history of heart or other current serious health problems. Participants were mostly unemployed (49%). Most participants were overweight with a mean ± SD BMI of 27.7 ± 8.4 kg/mal range . In tota2 and median benzene, ethyl-benzene, m+p-xylene, and o-xylene levels were significantly higher at the baseline visit compared to at the follow-up visit (p < 0.001) (The mean NOup visit . Personaup visit . Strong < 0.001) . No sign2 exposure was positively associated with blood pressure and negatively associated with blood vessel diameters. Each SD increment (4.96 µg/m3) in NO2 was associated with 2.42 mmHg  and 1.76 mmHg  increase in SBP and DBP, respectively  decrease in CRVE and −0.11 mm  decrease in mean baseline brachial diameter.NOectively . Each NO3) and o-xylene (2.51 µg/m3) were associated with a 2.07 mmHg  and 2.00 mmHg  increase in DBP, respectively (3) increment increase in benzene was positively associated with 24.88 µm (95% CI: 2.19 to 47.57 µm); p = 0.032) increase in cIMT  for each SD (3.12 ng/mL) increment in 3+4MHA  compared to international air quality standards, our participants’ personal exposure to NO2 and BTEX was relatively low, and (2) despite the relatively low exposure levels, we could demonstrate associations between air pollutants and several cardiovascular parameters: SBP and DBP (NO2 and BTEX), baseline brachial artery diameter (NO2), CRVE (NO2), and cIMT (benzene). The current study set out to determine personal air pollution exposure levels as measured by NO2 exposure concentrations (3–25.35 µg/m3) remained below the recommended WHO, European Union (EU) and South African air quality standards for NO2: annual exposure of <40 µg/m3 and 1-hour exposure of <200 µg/m3 [3–14.17 µg/m3) were higher than WHO and EU annual recommended standards (<5 µg/m3), although the median values were below the standards [The mean 7-day personal NOtrations  observed00 µg/m3 ,49,50,51tandards . 2 and median benzene concentrations at baseline in our study population (NO2: 13.6 ± 4.8 µg/m3 and benzene: 3.22 (0.77–14.17) µg/m3) were within the lower range of annual mean ambient NO2 and benzene concentrations of major European cities, with NO2 ranging from 8 µg/m3 in Stockholm to 43 µg/m3 in Barcelona, and benzene ranging between 2 and 12 µg/m3 across various cities [Furthermore, the mean NOs cities ,53,54. 2 levels in our study were comparable with recently reported annual mean NO2 levels from air quality monitoring stations in neighboring areas  [2 and individual BTEX concentrations in the current study were furthermore similar to those measured previously (2011–2014) in the Drakenstein sub-district, ~60 km from the City of Cape Town [2 and BTEX levels  were measured in more than 500 homes of families of African and mixed ancestry (NO2: 7.9 (Interquartile range (IQR): 3.8–13.3) µg/m3, benzene: 5.6 (IQR: 2.6–17.1) µg/m3, toluene: 19.8 (IQR: 9.3–53.2) µg/m3, ethyl-benzene: 2.1 (IQR: 0.9–53.2) µg/m3, m+p-xylene: 5.8 (2.4–16.2) µg/m3, and o-xylene 2.4 (IQR: 1.1–7.0) µg/m3).Compared to other studies from the Western Cape Province, the observed mean personal NO0 µg/m3) . The perape Town . In this3, respectively, toluene concentrations of 2.66 and 1.04 µg/m3, respectively, ethyl-benzene concentrations of 0.43 and 0.27 µg/m3, respectively, and xylene concentrations of 1.82 and 1.19 µg/m3, respectively,) were generally lower than the personal BTEX exposure levels measured in our study [Ambient outdoor BTEX levels are infrequently measured in the Western Cape Province of South Africa. The Provincial Government Report (2013) reported on BTEX levels (measured with passive samplers) from only two locations in one rural town  in the province . The repur study .2 and total BTEX (mostly driven by o-xylene) exposure was positively associated with blood pressure outcomes after adjusting for covariates  in a female population and showed that a 10 ppb increase in NO2 was associated with a higher pulse pressure (0.4 mmHg) [2.5 was also associated with higher SBP (1.4 mmHg), pulse pressure (1.0 mmHg), and mean arterial pressure (0.8 mmHg) [2 and vessel diameters as indicated by the baseline brachial artery diameter and CRVE. The vasoconstrictive effects of both long-term low-concentrations and short-term high-concentration exposure to ambient air pollution have previously been described [3 fine PM and 120 ppb O3) and a shorter exposure period (2 hour) compared to our exposure concentrations and exposure period [Our findings support those of Chan et al. (2015) who examined the effects of NO.4 mmHg) . In the .8 mmHg) . The aut.8 mmHg) ,61. Thisescribed ,63. In te period . The aute period .Previous studies have investigated the possible mechanism involved in air pollution exposure-associated vasoconstriction and suggested possible stimulation of the pulmonary vagal afferent neurons and the subsequent increase in sympathetic nervous system reflex activity or an upregulation (directly or via oxidative stress pathways) of vascular endothelin 1 and 3 (vasoconstrictors) ,64; howeBenzene exposure was associated with sub-clinical atherosclerotic changes as measured by cIMT in our study with a relatively large estimated effect on cIMT. cIMT is considered a marker of pro-atherosclerotic processes such as inflammation, a strong predictor of future cardiovascular events , and inc3+4MHA appeared to be a prominent urinary metabolite/tracer for exposure in our study as it strongly correlated with all personal exposure levels . 3+4MHA Xylene is a carcinogen and also associated with central nervous system abnormities such as brain and neurobehavioral morbidities ,72. In oThe results from the current study are presented with some strengths and limitations. Strengths of current study include the measurement of personal exposure levels, as opposed to levels obtained from centralized air quality monitoring stations in many previous studies from the SSA region. Personal measurements are generally considered a more accurate representation of exposure levels. An additional strength of the study is the fact that we used measurements of different cardiovascular endpoints to investigate physiological effects. To the best of our knowledge, this study is the first to explore the cardiovascular health effects of personal air pollution exposure, in combination with urinary exposure markers, in the South African research setting.Limitations of the study include a relatively small sample size representing only women, of whom the majority were smokers. The male participant enrolment rate was low, mostly due to employment obligations that resulted in difficulty to attend assessment visits. The high prevalence of smokers in our cohort may be ascribed to the high smoking rates that have previously been reported in the region ,74. The Our results show that personal ambient air pollution exposure in women residing in Cape Town, even at relatively low levels, is associated with markers of cardiovascular risk including blood pressure (SBP and DBP), vascular tone/diameter , vascular endothelial function (% FMD), and subclinical atherosclerosis (cIMT).