TWIST‐1 and DERMO‐1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high‐growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low‐growth capacity MSCs when assessed for ectopic bone‐forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application. Stem Cells2015;33:1878–1891This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age‐ and sex‐matched donors. Adherence to plastic was not indicative of potency, yet capacity for long‐term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high‐growth capacity or low‐growth capacity. Using this grouping strategy, high‐growth capacity MSCs were smaller in size, had greater colony‐forming efficiency, and had longer telomeres. Cell‐surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high‐growth capacity and low‐growth capacity MSCs, whereas STRO‐1 and platelet‐derived growth factor receptor alpha were preferentially expressed on high‐growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts  Potency assays that predict the therapeutic efficacy of bone marrow‐derived adult mesenchymal stem cells (MSCs) are critical to their successful application in regenerative medicine. Patients are still currently receiving cells of unproven potency, which in turn, may contribute to many of the suboptimal outcomes that are being reported However, despite a considerable body of research over the last decade, resulting in an increasing pace of investigational new drug (IND) submissions to the U.S. FDA, there is still no generally accepted definition of what constitutes a mesenchymal stem  cell The problem in establishing such benchmarks is that MSCs consist largely of heterogeneous subpopulations of adherent cells that are typically isolated from adult bone marrow that, when induced, are capable of self‐renewal, colony formation, and multilineage differentiation Another characteristic of MSCs gaining attention is the trophic factors they produce TWIST‐1 and DERMO‐1, and the expression of the cell‐surface biomarkers STRO‐1 and platelet‐derived growth factor receptor alpha (PDGFR‐α).Here, we sought to identify critical determinants of MSC potency by linking in vitro attributes such as self‐renewal and clonogenicity, cell size, telomere length, mRNA profile, and growth factor/cytokine secretion to in vivo tissue regeneration ability as determined by their ectopic bone‐forming ability. This study highlights that the minimum criteria suggested by the ISCT for defining MSCs are not in themselves predictive of stem cell potency. There is thus a need for common guidelines and protocols for the characterization of MSCs for clinical use, as suggested in the ISCT working proposal www.lonza.com) in maintenance media comprising of Dulbecco's Modified Eagle's medium (DMEM)  with 10% fetal calf serum , 2 mM l‐glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin , as described previously 6 cells in T75 flasks, allowed to adhere and form colonies, and stained with 0.5% crystal violet (Sigma‐Aldrich) after 14 days. Visible colonies were quantified only when they were greater than or equal to 50 cells, and not in contact with an adjacent colony. Efficiency was calculated by estimating the number of colonies formed per 105 cells plated.Human MSCs were isolated from the bone marrow of six healthy donors with no reported antibiotic sensitivity (Supporting Information Table S1) by plating human bone marrow mononuclear cell (MNC) fractions . After gating out Annexin V+ cells, a quadrant gate was applied to the live population, and the fraction of low forward scatter/side scatter (FSC/SSC) events recorded to obtain the relative percentage of small‐sized cells.Single‐cell suspensions of MSCs were stained with Annexin V, washed, and resuspended in MACS buffer  in PBS, pH 7.2) before analysis using FACSArray Bioanalyzer . Cells were replated at the same density and progressively subcultured for 13–15 passages  to estimate cumulative cell numbers and population doublings. Genomic DNA at different passages was isolated and telomere length analysis performed as described previously To assess MSC growth, cells were seeded at 5,000 cells per square centimeter and cultured under maintenance conditions. At 70%–80% confluence, cells were enzymatically removed with 0.125% trypsin/Versene and viable cells counted using Guava EasyCyte Plus  by gating at 2% of isotype control. For each marker and isotype, 20,000 events were acquired. All antibodies were purchased from BD Biosciences except IgM (μ‐chain specific)  and STRO‐1 .Single‐cell suspensions were stained with phycoerythrin‐ or fluorescein isothiocyanate‐conjugated anti‐human CD105 (266), CD73 (AD2), CD90 (5E10), CD45 (H130), CD34 (RAM34), CD49a (SR84), CD29 (MAR4), EGF‐R (EGFR.1), IGF‐IRα , NGF‐R , PDGFR‐α and β , CD11b (D12), HLA‐DR (TU36), CD19 (HIB19), CD14 (MфP9), CD106 (51‐10C9), CD146 (P1H12), SSEA‐4 (MC813‐70), STRO‐1 antibodies or the isotype‐matched controls IgG1κ (MOPC‐21), IgG2aκ (G155‐178), IgG2bκ (27−35), IgM, and IgG3 (A112‐3), and analyzed on a BD FACSArray Bioanalyzer and FlowJo software v8.0  and PBMCs were isolated by Ficoll‐Paque PLUS  density gradient centrifugation (400 x g for 30 min without brake). CD4+ T cells were isolated from PBMCs using EasySep negative selection human CD4+ T cell enrichment kit  and labeled with 5 µM carboxy‐fluorescein diacetate succinimidyl ester . CD4+ CFSE+ cells (0.5–1 × 105) were stimulated by anti‐CD3/anti‐CD28 MACSiBeads  at a ratio of four beads per T cell, to which MSCs were added at different T cell/MSC ratios and incubated at 37°C. Proliferation of CD4+ CFSE+ cells was measured after 7 days by flow cytometry and the percentage inhibition of T‐cell proliferation determined.All experiments using human blood were approved by the Institutional Review Board at the National University of Singapore (NUS‐IRB 09‐256) and comply with the Helsinki declaration. Written informed consent was obtained from all blood donors prior to blood collection. Adult human blood was collected in BD K2 EDTA Vacutainers  for ∼10 seconds. The conditioned media and salt wash were analyzed separately using Human Cytokine/Chemokine kit  for simultaneous quantification of 14 growth factors and cytokines, according to the manufacturer's instructions. Results are presented as logarithmic concentration for each analyte (growth factor or cytokine), which includes factors bound to matrix as well as secreted into media. Results are shown as mean of two individual experiments for each donor.www.mn-net.com) according to the manufacturer's instructions and quantified by NanoDrop 1000 . After conversion to cDNA , expression of mesodermal genes TWIST‐1 and DERMO‐1, osteogenic markers RUNX2, ALP, and BSP‐II, adipogenic markers PPARγ and CEBPα, and chondrogenic genes COL2A1 and SOX9 was assessed using TaqMan Gene Expression assays on an Applied Biosystems 7500 PCR System  (Supporting Information Table S2). CT values were normalized to β‐ACTIN and results plotted as relative expression units.For quantitative PCR, total RNA was isolated using a Nucleospin RNA II kit . The resulting purified biotin‐labeled complementary RNA (cRNA) was normalized and hybridized onto a HumanHT‐12 version 4 beadchip  using direct hybridization. The chip was then washed, blocked, and Cy3‐streptavidin bound to the hybridized cRNA. An Illumina BeadArray Reader using the Illumina BeadScan software was used to image the chip, and the image data converted into an expression profile by GenomeStudio (Illumina). After background subtraction, data were submitted to GeneSpring . The replicates were averaged and pairwise analysis performed, followed by a Student's t test with p < .05 and fold change ≥1.5. Two donor samples representing each group with two technical replicates were analyzed. The gene lists generated were uploaded using Entrez gene ID onto DAVID (david.abcc.ncifcrf.gov) for functional annotation clustering by GOTERM_BP_FAT with medium classification stringency p ≤ .05 were considered.For microarray, RNA extracted from MSCs was amplified using a TotalPrep RNA amplification kit according to the manufacturer's instructions . For the von Kossa and oil red O methods, the staining intensity values were normalized to DNA content. Results are expressed as relative intensity per microgram DNA . In these measurements, a darker stain implies a higher density value.Evaluation of the differentiation potential of MSCs for osteogenic, adipogenic, and chondrogenic lineages was performed as described previously www.medtronic.com) alone (control) and MASTERGRAFT Matrix with MSC from 6 individual donors  resulting in seven groups with 5 implants per group . The implants were randomly assigned to 18 mice, with up to 2 implants per animal. Donor MSCs at P4 (3 × 106 cells) were seeded onto scaffolds before implantation into subcutaneous pockets of 8‐week‐old immunodeficient mice , as described previously www.shimadzu.com) and a DÜRR MEDICAL‐CR 35 VET Image Plate Scanner DÜRR MEDICAL‐CR 35 VET Image Plate Scanner, Vancouver, WA, www.im3vet.com). At sacrifice (8 weeks), implants were removed, digitally photographed (Nikon D80), and imaged using a Skyscan 1076 µCT scanner  under parameters previously reported www.materialise.com) after applying appropriate threshold settings. After µCT analysis, implants were transferred to 10% neutral buffered formalin under vacuum for 1 week, decalcified, paraffin embedded, sectioned, and stained as previously reported www.takara-bio.com) and human osteocalcin . Sections were washed and bound antibody detected using the Immunopure ABC peroxidase staining kit as per the manufacturer's recommendations . In order to verify antibody specificity, staining with secondary antibody alone was performed on relevant control tissues . All stained sections were examined under a Zeiss AxioImager (Z1) upright microscope .Comparison of in vivo efficacy between individual MSC donors was assessed by ectopic bone formation assay. The experimental groups included MASTERGRAFT Matrix , unless otherwise stated, and p value < .05 was considered significant.Quantitative data were obtained in triplicates and reported as mean ± SD, unless otherwise stated. Statistical analyses were performed using Student's p < .001) . Significant donor variability in CFU‐F efficiency was observed, with donors A, C, and E having greater efficiency (7.4 ± 1.7) than donors B, D, and F (3.2 ± 1.1) (lo) and low side scatter (SSClo) lo/SSClo in quadrant 1, Fig. p < .001). Donors with higher CFU‐F efficiency also had a higher proportion of small‐sized cells , usually identified by flow cytometry as low forward scatter (FSCp < .05). Thus, we observed that changes in self‐renewal capacity only became evident upon long‐term passaging with MSCs from all donors having similar cumulative growth up to passage 6. Therefore, the high‐growth capacity MSCs seen here were also smaller in size and produced more CFU‐Fs than the low‐growth MSCs.Long‐term expansion without phenotypic change is a key parameter of MSC quality. We next monitored cell growth for extended periods 8–10 weeks) in culture. Notably, changes in cumulative growth revealed significant differences in the proliferative potential of MSCs from the different donors after seven passages  values than donors B, D, and F (low‐growth capacity), indicating that the high‐growth MSCs reduced their telomeres at slower rates than low‐growth MSCs. In addition, low‐growth MSCs failed to self‐renew after only 13 passages, whereas the high‐growth capacity MSCs continued to proliferate until at least 15 passages. Collectively, our data show that donor MSCs classified as having high‐growth capacity had an increased capacity for self‐renewal, a higher CFU‐F efficiency, and a larger proportion of small‐sized cells.As telomeres are tightly linked to cell division, we next assessed their status during the serial passaging of MSCs. Analysis from the first passage P1) until no further proliferation was observed (P13–P15) revealed progressive decreases in relative telomere length , antiscarring (HGF and FGF‐2), and antiapoptosis . All donor MSCs secreted detectable amounts of the various factors, albeit at varying amounts . Data from duplicate experiments for each donor show similar secretion levels for each of the factors tested and are presented as a mean value . Also, for each of these factors, no difference was observed between the high‐ or low‐growth capacity groups . However, secretion levels varied from <10 pg/ml for RANTES, MCP‐2, MIP‐1α, PDGF‐AA, and EGF; 10–100 pg/ml for LIF, PDGF‐BB, and fractalkine; 100–1,000 pg/ml for SDF‐1α, IL‐6, VEGF165, and HGF; and >1,000 pg/ml for MCP‐1 and FGF‐2 (Supporting Information Table S3). It is to be noted that the analysis of growth factor and cytokine secretion were performed using cells at passage 4 at which stage the levels of secretion between the two groups were not significantly different. At this passage, we did not observe significant differences in the growth capacity between the two groups, as can be seen from their cumulative cells numbers , population doubling, PD , and cumulative PD  .Some of the regenerative capacity of MSCs has been attributed to the factors they secrete when administered to treat disease or injury (see review TWIST‐1 and DERMO‐1. High‐growth capacity MSCs demonstrated significantly higher (p ≤ .05) transcript levels of these markers  in high‐growth capacity group (Supporting Information Table S4A), which are indicative of a faster rate of turnover. Adhesion to the matrix and neighboring cells correlates with cell cycle progression Gene ontology analysis by DAVID functional annotation clustering RUNX2, BSP‐II, ALP, COL2A1, SOX9, CEBPα, and PPARγ under noninduced conditions by qPCR  compared with both scaffold alone or scaffold seeded with low‐growth capacity MSCs, as determined by µCT analysis , cumulative cell number , STRO‐1 expression , mRNA level of TWIST‐1 , and mRNA level of DERMO‐1 . The regression plots are provided as Supporting Information Figure S7.Taking into consideration all the results above, we identified the following in vitro parameters that positively correlate with in vivo bone formation: cell size (To date, no systematic correlation between the properties of MSCs in vitro and their in vivo efficacy has been demonstrated, primarily because there is no universally accepted in vitro method for predicting the therapeutic potency of MSCs. A key study attempting to provide clinically relevant potency assays demonstrated that the doubling time of MSCs correlates with in vivo bone formation By comparing our in vitro findings with in vivo bone formation ability, we conclude that MSCs with higher CFU‐F efficiencies, a larger proportion of small‐sized cells, and enhanced growth capacity were able to form increased amounts of bone at ectopic sites. Another important outcome from this study was the need to include additional cell‐surface biomarkers in the assessment for the identification of MSC, thus highlighting that the ISCT panel of surface markers did not adequately predict the in vivo potency of MSCs. Notably, in this study, the use of additional surface markers such as STRO‐1 and PDGFR‐α was able to distinguish between MSCs with high versus low ectopic bone‐forming ability, and thus may help to identify more potent MSCs. Importantly, the expression of STRO‐1 on bone marrow‐derived MSCs has been extensively studied Another feature of MSCs pertains to their immunomodulatory activities. These cells appear to have potent immune regulatory functions that make them good therapeutic candidates for tissue injury and/or inflammation TWIST‐1 and DERMO‐1, mRNA transcripts shown by Psaltis et al. In addition to phenotypic and functional assays of MSCs in this study, a global gene expression analysis was performed. Results revealed the enrichment of proliferation‐associated processes in high‐growth MSCs and maturation‐related processes in low‐growth MSCs, which together suggest a physiological basis that may underpin their phenotypic divergence in vitro. As the MSCs were derived and propagated from donors according to standardized procedures, differences in the physiology of the cells might be due to an underlying variation in their transcriptome. Also, increased expression of There is a growing acceptance that the tissue regeneration capacity of MSCs is driven, in part, by the factors they secrete when transplanted to sites of injury or disease Although both groups of MSCs secreted similar levels of growth factors and cytokines and showed comparable capacities for osteogenic differentiation in vitro, they produced varying amounts of ectopic bone in vivo. This could be because the high‐growth MSCs were able to survive longer and have sustained growth because of relatively longer telomeres and a higher proportion of clonogenic small‐sized cells. The deposition of bone in the scaffold suggests that the capacity to deposit a mineralized matrix is spatially controlled, and that the most adaptable MSC to achieve this may be the high‐growth capacity cells. That the standard in vitro osteogenic assay yields a poor prognosis is in accord with a previous study from our group and others showing that ex vivo matrix mineralization assays lack specificity, and shows little or no concordance with true bone formation Despite the immense amount of work done over the last decade, MSCs are still a relatively poorly understood, heterogenous mixture of cells with unpredictable properties. As pointed out by Mendicino et al. in their recent review We have identified the MSC characteristics of cell size and in vitro growth capacity, together with expression of STRO‐1, TWIST‐1 and DERMO‐1 as being positively correlated with ectopic bone‐forming ability of implanted MSCs. We thus advocate the use of such additional criteria when assessing MSC quality before applying them as a cellular therapy.R.M.S.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; B.R.: collection of data and data analysis and interpretation; M.R.: data analysis and interpretation and manuscript review; J.H.H.: financial support, provision of study material, and manuscript review; K.J.P.: experiment design, collection and assembly of data, data interpretation, and manuscript review; O.R.: provision of study material, data analysis and interpretation, and manuscript review; P.S.: collection and assembly of data, data analysis and interpretation, and manuscript writing; L.W.S.: conception and design, provision of study material, data analysis and interpretation, and manuscript review; V.N.: conception and design, financial support, data analysis and interpretation, and manuscript writing; S.M.C.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing, and final approval of manuscript.The authors indicate no potential conflicts of interest.Supplementary Information Figure 1 R2Click here for additional data file.Supplementary Information Figure 2Click here for additional data file.Supplementary Information Figure 3Click here for additional data file.Supplementary Information Figure 4Click here for additional data file.Supplementary Information Figure 5Click here for additional data file.Supplementary Information Figure 6Click here for additional data file.Supplementary Information Figure 7Click here for additional data file.Supplementary Information DataClick here for additional data file.Supplementary Information LegendsClick here for additional data file.


Eisenia fetida were allowed to grow in presence or absence of MCs  for 1, 7, and 14 d. Results showed that MCs could bioaccumulated in earthworm. A stimulatory effect on catalase and glutathione oxidase activities induced by MCs was found on day 1, and both of them were significantly inhibited at 100 and 1000 μg kg−1 on days 14. The superoxide dismutase activity was relatively insensitive. Significant increase of malondialdehyde content and decrease of neutral red retention time were observed at 100 and 1000 μg kg−1 on days 7 and 14. Our results suggest that MCs induces oxidative stress on earthworms, which leads to disruption of the antioxidant system and lipid peroxidation, as well as alterations in lysosomal membrane stability.Irrigation with eutrophic water containing cyanobacteria toxins poses a potential risk to soil animals. To evaluate ecotoxicological effect of microcystins (MCs) on earthworms, filter paper acute toxicity test, avoidance test and a 14-d artificial soil test were carried out. No acute toxicity was found in the filter paper test, and earthworms showed no avoidance response to MCs exposure. In the artificial soil test,  MCs exposure induced thyroid endocrine disruption and transgenerational effects of developmental neurotoxicity in zebrafish offspring as well as in embryo4. More than 200 different structural analogues of MCs, with a range of molecular weights from 882 to 1116 Da, have been identified from cyanobacterial blooms and cultures6. Microcystin-leucine-arginine (MC-LR) is found to be the most common and potent analogue, followed by Microcystin-arginine-arginine (MC-RR) and Microcystin-tyrosine-arginine (MC-YR)7. With the increasing prevalence of these cyanobacteria blooms, more attention is being paid to impacts caused by MCs.In eutrophic fresh water lakes, a group of toxic compounds are produced by some bloom-forming cyanobacteria. The most prominent of these cyanotoxins are microcystins (MCs). Owing to their potential carcinogenicity, MCs can negatively affect both public health and fundamental ecological processes11. Total MC concentrations in surface waters varied from less than 1 μg L−1 to 29, 000 μg L−1 16. However, high concentrations of MC would be from very dense cyanobacterial biomass, and the concentration of MCs in most of the water samples were less than 100 μg L−1 19. Although the MCs concentration in field samples collected near Dianchi Lake were relatively low, ranged from 1.43 to 21.3 μg kg−1 20, high concentrations of MC will occur by irrigation with water containing dense cyanobacterial biomass. Moreover, once come in contact with soil, MCs can persist in soils with a half-life ranging from several days to several months22, depending on the efficiency of degradation, mainly photolysis and bacterial degradation. Studies on the absorption of MCs in soils are rare. It is suggested that organic carbon content and clay content in soils were important for the adsorption of MCs, and sandy soil was incapable of the removal of MCs24. However, Eynard et al.25 indicated that soil was unable to protect groundwater from surface water that contains cyanotoxins. Therefore, there is a possibility that soils adjacent to lakes and reservoirs may be contaminated by MCs. And the low absorption of MCs in soils could lead to their high bioavailability to soil organisms, animals and plants. However, very few studies have examined the impact of MCs on soils, especially on soil animals, which play important roles in the soil ecosystem. A recent study reported the toxic effects of MC-LR on earthworms26. In their study, MC-LR caused reproductive, biochemical, and cellular toxicity to earthworms. Li et al.27 used the nematode Caenorhabditis elegans as a model animal for assessing the toxicity induced by microcystin-LR. Reduced life span, extended generation time, decreased brood size, delayed development and suppressed locomotion behavior were found in C. elegans after exposed to MC-LR. Caenorhabditis elegans were also used for a chemotactic behavior study induced by MCs28. They found that MCs altered AWA but not AWC sensory neuron function via mechanisms other than protein phosphatases inhibition. However, the survival or reproduction of springtail Folsomia candida was not affected by water bloom biomass at concentration up to 4 g kg−1 DW soil29. As it is known to us, earthworms play vital roles in soil system, such as nutrient cycling, climate, water purification, remediation and restoration30. Moreover, earthworms can be used as bioindicators of various soil contaminations due to their sensitivity to pollutants32. Antioxidant enzymes in earthworms, which protect cells against adverse effects of reactive oxygen species (ROS), are used as indicators for evaluating the environmental effect of soil pollutants35. As a sensitive cellular biomarker, lysosomal membrane stability can provide valuable information on cellular damage36. Among earthworms, Eisenia fetida is sensitive to various toxicants and can be cultured easily under laboratory conditions. So it has been used as a standard animal for toxicology experiment37. In the present study, E. fetida was used for assessing the toxicological effects of MCs. In addition, biochemical responses in earthworms can be used as early warning indicators of soil contamination.MCs can be brought into contact with soils by irrigation source water that contains cyanobacteria; in addition, cyanobacterial blooms can be applied directly to soil as organic fertilizer after being intentionally harvested from lakes16, India38, Australia39, United States40 and Finland12. Consequently, it is possible that large areas of farmland near contaminated lakes are exposed to irrigation source water containing cyanobacteria blooms. In the present study, we measured mortality, avoidance responses, bioaccumulation, biochemical responses, and lysosomal membrane stability in earthworms exposed to different concentrations of cyanobacteria crude extract. These findings help to give a better understanding of the potential environmental risk of irrigation with cyanobacteria bloom into agricultural soils.Cyanobacterial blooms have become more frequent in recent years in many large freshwater lakes and reservoirs. Surface water containing MCs used as irrigation source were reported all over the world, including but not limited to ChinaThere was no significant effect of microcystins on earthworms with filter paper test. Survival was 100% in all treatments after the 72 h exposure. In addition, earthworm weight was not significantly affected by MCs concentration , nor were morphological abnormalities observed in any of the treatments.Earthworm net avoidance response was not significantly affected by MCs treatment in soils. .−1 treatments. For 10, 100 and 1000 μg kg−1 treatments, MCs were not detected on day 1, but increased along with increasing MCs dose on days 7 and 14 .There was no evidence of MCs bioaccumulation in earthworms from control and 1 μg kg−1 did not affect CAT activity regardless of exposure time. However, CAT activity responded quickly to the application of higher concentrations of MC; CAT activity at 10, 100, and 1,000 μg kg−1 increased significantly after 1 d  used in their study is smaller than ours (~500 mg), and MCs are more toxic to younger earthworms. Toxic effects of MCs on aquatic animals have also been reported before. Oberemm et al.41 found a dose-dependent increase of mortality rate of chub  after exposure to 0.5, 5, or 50 μg MC-RR or MC-LR L−1. Liu et al.42 also reported mortality (LC50 = 593.3 μg/L) of juvenile loach (Misguruns mizolepis) after exposure to 1, 3, 10, 100, or 1,000 μg MC-LR L−1 after 7 d. Various abnormalities, as well as dose- and time-dependent survival rate was found after micro injection of 300, 750, and 900 nM MC-LR into zebrafish (Danio rerio)43. However, some animals such as mollusks showed tolerance to cyanotoxins. Vasconcelos44 reported that less than 1% mortality of mussel  was found after fed on a toxic M. aeruginosa strain for 16 days. During this period mussels accumulated up to 10.5 µg g−1 MCs dry mussel weight. Anodonta cygnea was also found to be able to accumulate high levels of microcystins produced by Oscillatoria agardii HII without any visible damage45. There are no specific carriers that carry MCs, which are relatively lipophobic, into hepatopancreatic cells46. Moreover, MCs used in the present was cyanobaterial extract contained mainly MC-RR, which is less toxic than the most common congener MC-LR in a mice test47. Thus, toxicity of different MC congeners in earthworms should be explored in the future. Besides, in view of the toxicity of MCs to aquatic animals, short- and long-term effects of MCs on other soil organisms should also be evaluated. Our results showed that concentrations of MCs in earthworms were high when MC concentrations in treatments were above 10 μg kg−1, suggesting that MCs may bioaccumulate in earthworms. With less microbes and no light in the artificial condition, which are main factors for MCs degradation1, MCs degradation in artificial soil is much lower than that in natural soil. Degradation of MCs in the artificial soil is less than 20% at the end of the exposure experiment. So the bioaccumulation of MCs in earthworms in natural soil should be re-evaluated due to different soil type and different quantity of MCs. Moreover, we were unable to completely remove earthworm gut contents, even when gut contents of earthworms were voided with wet filter paper for 24 h after exposure. Hence, we suggest that the biological responses (such as response of the antioxidant system) observed in our study are better indicators of impacts from MCs than tissue levels.In this study, we found no evidence that MCs are acutely toxic to 48. When cellular antioxidant defense fail to remove excessive ROS, oxidative stress results in organisms49. CAT, which eliminates hydrogen-peroxide, plays an important role in anti-oxidant systems. In our study, CAT activity in E. fetida was sensitive to MCs exposure. A significant increase was found at 10, 100, 1,000 μg kg−1 after only 1 d exposure, indicating that MCs lead to oxidative stress in this earthworm species50. However, the stimulatory effect of 100 and 1,000 μg kg−1 in our study disappeared after 7 d, with a significant decrease observed after 14 d exposure. The same time-dependent effect was found in crabs, Pinho et al.51 showed that CAT activity increased after exposed to 5.32 mg MCs kg−1 day−1 for 2 days, whereas decreased at day 7. A possible explanation is that the production of H2O2 at the early stage may induce an increase of CAT activity, whereas the natural antioxidant defenses may have become saturated as incubation time lengthened, resulting in accumulation of ROS in earthworms, thereby inhibiting CAT activity53. As antioxidant system vary with animal species, different result was found in freshwater clam Diplodon chilensis patagonicus, in which a continuous stimulatory effect of CAT was observed after exposed to an estimated mean weekly dose of 0.625 mg MCLR g−1 clam for six weeks54. Leão et al.55 found decreased CAT activity in an estuarine worm (Laeonereis acuta) after 48 h exposure to 2 mg mL−1 lyophilized cells of toxic M. aeruginosa. Moreover, we detected all the biochemical indicators with whole earthworm tissue with no distinction of different organs. Lower CAT level was found in gills than other organs (liver and kidney) of tilapia56. So this may raise questions on the difference of antioxidant response in different organs. Therefore, alternation of antioxidant enzyme activities in different earthworm organs after exposed to MCs should be studied in the future.The biochemical indicators varied with doses of MCs and duration of exposure. One of the toxicity mechanisms of MCs to animals is oxidative stress2O2 in organisms by using reduced glutathione as a hydrogen donor. Similar to CAT activity, a stimulatory effect was observed at 1,000 μg kg−1 after 1 d exposure, and a significant inhibition at 100 and 1,000 μg kg−1 began after 7 d exposure, which continued through day 14. Stimulatory effect of GPx activity was also observed in embryos of zebrafish exposed to 0.5 μg L−1 MC-LR57, and in hepatocytes of C. carpio exposed to 10 μg L−1 MC-LR58. However, GPx activity was increased in liver and intestine of Corydoras paleatus exposed to 2 μg L−1 MC-RR, whereas strongly inhibited in gills at all tested concentrations59. Same differential impact vary with organs was found in tilapia56. Similar to CAT, as previously discussed, antioxidant response in different earthworm organs should be re-evaluated. Interestingly, same as our results, a previous study on earthworms60 also found that GPx activity was induced by naphthenic acids after 1 d but inhibited after 14 d of exposure. The reasons might be that (i) GPx activity was inhibited by excessive ROS in cells; and (ii) large amounts of glutathione (GSH) were consumed to remove ROS.GPx is another enzyme that can remove H·superoxide anion radical (O2−) in the cell is transformed to H2O2 by SOD and then H2O2 is transformed to harmless H2O and O2 by CAT and GPx under oxidative stress62. Unlike CAT and GPx activity, no significant change was found after 1 d in our study. A stimulatory effect was found only at 100 μg kg−1 after 7 d, which probably was caused by O2− accumulation51. Inhibition was found at 1,000 μg kg−1 after 14 d. The possible reason is that hydrogen peroxide, peroxyl radicals and singlet oxygen induce inactivation of SOD or the highly reactive superoxide was eliminated66. Increase as well as decrease of SOD activity in organs after treated with MCs were found by previous studies69. Similar to CAT and GPx activities, SOD activity varies with animal species.SOD also plays a vital part in eliminating ROS. The 70. Various contaminants are known to induce lipid peroxidation due to excessive ROS71. In our study, MDA content in earthworms was significantly higher than that observed in controls after 7 d and 14 d of exposure to higher concentrations of MC . These results suggest that MCs lead to lipid peroxidation and induce oxidative injury in earthworms.MDA results when unsaturated fatty acids react with free radicals in cellular membranes. It is used frequently as a sensitive indicator that reflects lipid peroxidation and indicates intracellular injury48. Oxidative stress caused by excessive ROS production was found in several MC-treated aquatic and terrestrial organisms, and an increase in lipid peroxidation was found as well51. Regarding plants, SOD activity in rape (Brassica napus L.) decreased as toxin concentration increased, while activity was stimulated in rice (Oryza sativa L.)72. Stimulatory effects of MCs on SOD and GPx activities also were found by Chen et al.10 and Gehringer et al.73 in apple trees  and garden cress (Lepidium sativum), respectively. Oxidative stress was found in several MC-treated animals and plants, changes in antioxidative activity varying with time and tested species.Oxidative stress in animals and plants caused by MCs has been reported in recent years. Generation of ROS and disruption of mitochondrial electron transport chain were found in MC-treated rats74. LMS is an early indicator of various environmental contaminants, and LMS in earthworm coelomocytes is often evaluated by the NR retention assay75. In the present study, high concentrations of MC significantly decreased LMS in earthworm coelomocytes. A same inhibition effect of NRRT was reported by Wen et al.26 in MC-LR treated earthworms. However, the mechanism causing alternations in LMS is still unknown. Evidence suggests that ROS produced in the cellular system affects various cellular organelles77. Consequently, generation of ROS induced by MCs may be the cause of alterations in LMS. This putative mechanism should be explored in future studies to better understand the impact of MCs on LMS on earthworm coelomocytes.Our results demonstrated that MCs can induce oxidative stress in earthworms, as observed in other animals and plants, which can lead to the disruption of the antioxidant system and lipid peroxidation. Nevertheless, the duration of our study was only 14 d; we recommend that earthworm responses to MCs be evaluated over longer time periods and in natural conditions. Generally, impaired lysosomal reactions precede cell and tissue pathology; therefore, lysosomal systems can respond quickly to pollutant exposure78. Although the composition of artificial soil was set to mimic natural soil, the microbial composition of artificial soil is much different from that in natural soil. Moreover microorganisms play important roles in degrading MCs in soils. The implication for natural soil systems is unclear, so field experiments are needed in the future.Soils irrigated with cyanobacteria-containing water may suffer varying degrees of MCs contamination, which could pose a threat to earthworms in the soil. Although no significant effect on earthworms was observed at low MC concentrations treatment, effects on earthworms after long-time and/or repeatedly contact with MCs are still unknown. In the artificial soil test, earthworms are cultivated in artificial soil according to OECDEarthworms were purchased from a local earthworm farm in Nanjing. Adult earthworms weighing about 500 mg with well-developed clitella were selected. To void gut contents, earthworms were placed on damp filter paper in Petri dishes for 24 h before testing.et al.79. Cyanobacterial cells obtained from Dianchi Lake (~1 g DW) were homogenized with 25 mL of 5% (v/v) aqueous acetic acid after freeze-drying. The homogenate was subjected to an ultrasonic bath for 5 min, and then centrifuged at 10,000 r min−1 at 4 °C for 15 min. After re-extracting the residue two more times as before, all the supernatant was collected and then applied to 5 g HLB extraction cartridge. The cartridge with toxin was rinsed with 50 mL of 5% (v/v) aqueous methanol. Subsequently, the cartridge was eluted with 100 mL 100% aqueous methanol. The eluant was evaporated to dryness, and then 10 mL deionized water was used to dissolve the toxin. Combined toxin-containing solutions were stored at −40 °C before use. According to Corbel et al.80, microcystin variants were analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS), using a Waters Acquity UPLC system coupled to a triple-quadruple mass spectrometer  via an electrospray ionization (ESI) interface. MC-LR (14.92 mg L−1), MC-YR (5.90 mg L−1) and MC-RR (61.61 mg L−1) were found in the extract after the analysis. The toxin extract was diluted as needed for subsequent use.In the present study, we used a natural cyanobacterial bloom extract so as to mimic natural toxic conditions with multiple MCs. In order to prevent interference from impurities, the extract was purified with an Oasis HLB extraction cartridge (Waters). The method of extraction and purification was modified from Harada 81. Considering there are no previous studies reporting the toxicity of MCs on earthworms, a wide range of MC concentrations was used for the acute toxicity test . After placing single pieces of filter paper on the bottom of Petri dishes, 1 mL of each MC treatment concentration or deionised water (controls) was added to wet the entire filter paper. Then one earthworm was randomly placed in each Petri dish. Five replicates were used for each treatment. The Petri dishes were incubated in the dark at 20 ± 1 °C for 72 h and the mortality was recorded every 24 h; earthworm weight and morphological changes also were observed.The acute toxicity test was conducted according to OECD guidelines82, avoidance tests were performed in plastic containers with three replicates for each treatment. Soils used in the test was collected (0–15 cm) from an area adjacent to Lake Taihu at Suzhou, with no known previous history of MCs application. Some of the physicochemical properties are as follows: 27.9% sand, 31.0% silt, 41.2% clay, pH 7.46, 9.91 g organic matter kg−1 soil, 0.78 g total nitrogen kg−1 soil, 15.4 mg available P kg−1 soil, 150 mg available K kg−1 soil. After taken back to the laboratory, the soil samples were homogenized and passed through a 2-mm sieve to sift out roots and other large debris. The soil samples were used for physico-chemical analysis after air-dried at 25 °C in 48 h. The remainders were kept fresh at 4 °C for avoidance test. Contaminated soil was produced by thoroughly mixing soil and MCs containing deionized water with final treatment concentrations of 1, 10, 100, 1,000 μg kg−1 dry soil. Control soil received an equal amount of deionized water. All treatments were adjusted to 60% of maximum water-holding capacity. Briefly, the container was filled half with contaminated soil and half with control soil at each end. After placing ten earthworms on the soil surface along the transition line between control and contaminated soil in each test container, perforated plastic films were used to cover the containers. The containers were incubated in the dark at 20 ± 1 °C for 48 h. The numbers of earthworms in the control vs. contaminated sections of each container were recorded after incubation. The net avoidance response (NR) was determined with the following formula: NR = (C − T)/N × 100%, where C, T, and N represent the numbers of earthworms in the control soil (C), the contaminated treatment soil (T), and the total number of earthworms (N), respectively.According to ISO guideline78 guidelines. The artificial soil was composed of 10% finely ground sphagnum peat, 20% kaolin clay and 70% industrial sand with 35% water content and 7.0 ± 0.2 pH value. Before the test, earthworms were acclimated for one week in untreated artificial soil at a controlled temperature of 20 ± 1 °C with 80–85% relative humidity under 400–800 lux of continuous light. Different amount of MCs were added in the soil to make a final MC concentrations of 1, 10, 100, 1,000 μg kg−1 dry soil. The control soil received an equivalent amount of distilled water. The MCs concentrations in the artificial soil prior to the exposure were 0.65, 8.47, 90.1 and 958.3 μg kg−1 dry soil. Twelve earthworms were added to each 1 L wide-mouth bottle filled with 500 g contaminated soil. Each soil treatment included three replicates. The bottles were sealed with porous plastic film, allowing exchange of air, and incubated at 20 ± 1 °C with 80–85% relative humidity under 400–800 lux of continuous light for 14 days. Four earthworms were randomly collected on days 1, 7, and 14 following application of MCs. Prior to analysis, the gut contents of all the earthworms were voided as mentioned above. No mortality was observed during the experiment.A 14-day toxicity test was performed in artificial soil according to OECD83. The limit of detection (LOD) for MC-LR, MC-RR and MC-YR are 0.022, 0.024 and 0.030 μg kg−1, respectively. The recovery rate ranged from 83.5% to 97.5% (RSD ranged from 0.1%–2.3%). Briefly, lyophilized earthworms were homogenized with 10 mL of BuOH:MeOH:H2O  after ground with a mortar. The homogenate was subjected to an ultrasonic bath for 3 min, and then centrifuged at 4,000 r min−1 at room temperature for 20 min. After re-extracting the residue two more times as before, all the supernatant was collected and then applied to 500 mg Oasis HLB extraction cartridge. The cartridge with toxin was rinsed with 20 mL of 5% (v/v) aqueous methanol. Subsequently, the cartridge was eluted with 15 mL 100% aqueous methanol. The eluant was evaporated to dryness, and then 2 mL 100% aqueous methanol was used to dissolve the toxin. The dissolved toxin was then applied to a silica gel cartridge (2 g)/plus silica gel (0.69 g) tandem cartridge. The cartridge with toxin was rinsed with 20 mL of 100% aqueous methanol, and eluted with 20 mL 70% aqueous methanol. The eluant was evaporated to dryness and the residue was dissolved in 0.1 mL 100% aqueous methanol. The toxin solutions obtained were stored at −40 °C for UHPLC/MS detection.Earthworms were lyophilized after voiding gut contents. MCs in lyophilized earthworms were determined as described by Xie and Parket al.20 with slight modification. The LOD for MC-LR, MC-RR and MC-YR are 0.25, 0.25 and 0.50 μg kg−1, respectively. The recovery rate ranged from 54.9% to 97.4% (RSD ranged from 4.3%−16.9%). Briefly, lyophilized soil samples were extracted 3 times with 30 mL of 0.1 M EDTA-0.1 M Na4P2O7 with 10 min ultrasonic bath after ground with a mortar. The homogenate was then centrifuged at 4,000 g for 10 min. After turning the pH value of the supernatant to pH 3 with TFA, the solution was centrifuged again with the same condition. Then the aqueous extractions were applied to an Oasis HLB extraction cartridge. The cartridge with toxin was rinsed with 15 mL of 20% (v/v) aqueous methanol, and eluted with 10 mL 90% aqueous methanol. The eluant was evaporated to dryness and the residue was dissolved in 0.1 mL 100% aqueous methanol. The toxin solutions obtained were stored at −40 °C for UHPLC/MS detection.The artificial soil was lyophilized after collected at the end of the test. MCs in soils were determined as described by Li 84. Earthworms were ground under ice-cold conditions in 0.85% NaCl solution  with a prechilled mortar. The homogenate was centrifuged at 3,000 rpm at 4 °C for 10 min. The supernatants were used for various analyses. Several test kits  including Catalase (CAT) assay kit (Ammonium molybdate method), Glutathione Peroxidase (GPx) assay kit (Colorimetric method), Total Superoxide Dismutase (T-SOD) assay kit (Hydroxylamine method), Total protein quantitative assay kit and Malondialdehyde (MDA) assay kit (TBA method) were used to measure CAT activity, GPx activity, total SOD activity, protein content and MDA content, respectively.Enzymes were extracted according to Mishra and Dash85. The extrusion medium  was used to rinse earthworms. Coelomocytes secreted in the medium were washed three times with 100 mM PBS (pH 7.3) to remove mucous. Neutral red (NR) retention assay was used to evaluate lysosomal membrane stability of coelomocytes as described by Lowe and Pipe86.The non-invasive extrusion method was used to obtain earthworm coelomocytesSPSS software  was used for statistical analyses. Data were expressed as mean ± SD. One-way ANOVA followed by the Duncan test were used to test for differences between different exposure levels and the control. Means of biochemical indicators were compared by two-way ANOVA with dose and duration treatments as independent variables. The criteria for significance were set at p < 0.01 and p < 0.05.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).The results obtained from various tests and multiple biomarkers lead us to conclude that: (1) MCs do not induce acute toxicity in earthworms; (2) earthworms showed no avoidance response to MCs exposure; (3) MCs were bioaccumulated in earthworms after exposed to high concentrations of MCs; and (4) MCs induced oxidative stress in earthworms, which lead to disruption of the antioxidant system and lipid peroxidation, as well as alterations in LMS. More studies are encouraged to figure out the thorough impact of MCs on soil animals.


LTPs) are important causes of plant‐food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract‐based diagnosis is often complicated by co‐sensitization to Bet v 1, the major birch pollen allergen, its cross‐reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant‐derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant‐food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.In the Mediterranean area, lipid transfer proteins ( Protein identification was performed as described by Hemmer et al.ImmunoCAP IgE‐inhibition experiments were carried out as described in Method 3Phleum pratense) were determined by ImmunoCAP, ImmunoCAP ISAC, and/or ELISA. Interestingly, ten of the patients had IgE antibodies to Pru p 3 (76.9%), nine (69.2%) to Bet v 1, and only two (15.4%) to profilin  showed bands at the molecular weight corresponding to LTP in the extracts of raspberry, apricot, and peach (a representative immunoblot of patient 2 is shown in Figure there is a benefit of using cooked extracts for prick to prick tests in the diagnosis of LTP sensitization as also suggested by Asero et al.in all of our ten LTP‐sensitized patients, raw and cooked extracts from peach showed strongest IgE reactivity, suggesting that Pru p 3 carries most of the epitopes recognized by patients' IgEs.Even though the primary LTP‐sensitizing source is also not always known in Mediterranean countries, the majority of LTP‐sensitized patients in this area react with Pru p 3 and Pru p 3 usually initiates the LTP‐allergy syndrome.4We have established that nsLTPs play an important role as plant‐food allergens in a Central European population reacting with strong symptoms. Patients from the central and northern parts of Europe, where primary sensitization to Bet v 1 usually occurs via the respiratory tract, often present only with mild symptoms after consumption of plant‐food material. In these subjects, plant‐derived food allergy is mediated by Bet v 1‐related proteins causing allergic cross‐reactions due to the high sequence similarity with Bet v 1. In contrast, allergic reactions to plant‐derived food in Southern Europe are usually associated with systemic reactions, due to a primary sensitization to LTPs, very potent allergens, usually causing severe reactions.However, severe reactions to fruits, vegetable, and storage plants do also occur in Northern and Central European individuals. This demands a careful, diagnostic workup scheme for allergic patients, which fulfills the needs to distinguish between different highly cross‐reactive allergens. Bet v 1‐related allergens, profilins, and the nsLTPs are widely distributed throughout the plant kingdom, limiting the use of crude plant extracts for differential diagnostic purposes.A major drawback of CRD is that the clinical relevance of the marker allergens requires a clinical validation and marker allergens should be recognized by the majority of the patients in each geographical area. As all Austrian LTP‐sensitized patients screened for specific IgEs to peach and to plants representative of a Central European Cuisine displayed strongest IgE reactivity to peach extract, we conclude that Pru p 3 can be used as the marker allergen for LTP sensitization also in Central Europe.Rosaceae family, it might be impossible to identify the LTP‐sensitizing source in Central Europe.The fact that extracts from peach and from apricot inhibited IgE reactivity to rPru p 3 similarly showed that due to the high sequence similarity between LTPs within the The authors declare no conflict of interests regarding the publication of this article.N. Mothes‐Luksch, M. Raith, G. Stingl, S. Wöhrl, and I. Swoboda designed the research. N. Mothes‐Luksch, M. Raith, M. Focke‐Tejkl, E. Razzazi‐Fazeli, and R. Zieglmayer performed the experiments. N. Mothes‐Luksch, M. Raith, S. Wöhrl, and I. Swoboda wrote the manuscript. All authors read and approved the final manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Moreover, PBQS also demonstrates excellent long‐term cycling stability  and superior rate capability  in Li battery. Besides the exciting battery performance, investigations on the structure–property relationship between benzoquinone (BQ) and PBQS, and electrochemical behavior difference between Li–PBQS battery and Na–PBQS battery, also provide significant insights into developing better Li‐organic and Na‐organic batteries beyond conventional Li‐ion batteries.In concern of resource sustainability and environmental friendliness, organic electrode materials for rechargeable batteries have attracted increasing attentions in recent years. However, for many researchers, the primary impression on organic cathode materials is the poor cycling stability and low energy density, mainly due to the unfavorable dissolution and low redox potential, respectively. Herein, a novel polymer cathode material, namely poly(benzoquinonyl sulfide) (PBQS) is reported, for either rechargeable Li or Na battery. Remarkably, PBQS shows a high energy density of 734 W h kg For a conjugated‐carbonyl‐based organic cathode material, the redox potential is usually between 1.5 and 3.0 V versus Li−1 = ≈1339 W h kg−1) and good availability for practical use. However, due to the high reactivity of benzoquinone monomers, the polymerization is rather difficult and no chemical synthesis of such polymers with high benzoquinone density was reported as far as we know. Herein, based on our understanding on the polycondensation reaction between halogenated quinone and sulfide,2PDHBQS−1, but also greatly elevate the redox potential by about 0.6 V. As a result, PBQS shows a remarkable improvement in energy density (2.67 V × 275 mA h g−1 = 734 W h kg−1) of Li‐organic battery. Due to the specialty of conjugated‐carbonyl and the elevated redox potential, PBQS can be also directly applied as cathode for Na‐organic battery,−1 = 557 W h kg−1) than those of most inorganic cathodes for Na‐ion batteries.The structure diversity of organic materials encourages us to develop organic cathode materials with both higher energy density and better cycling stability. Our strategy is to design and synthesize insoluble quinone‐based polymers possessing both high redox potential and high theoretical capacity. Benzoquinone (BQ) unit is one of the best choices on building blocks because of its superiority in both aspects22.1Scheme2S) to monomer , solvent and temperature, we proposed a new polymerization reaction between DCBQ and Li2S as shown in Scheme 2S to synthesize PAQS , preventing them from further reduction by sulfide anions. Due to the relatively low polymerization degree, the viscous mixture is troublesome to treat directly, which was acidified by HCl to separate the product, since such kind of phenolate tends to precipitate in acid solution.The structure of PBQS Scheme1b was Various characterization methods were applied to confirm the structure of PBQS. First, elemental analysis was conducted for a preliminary estimation. The measured C, H, O, S, N, and Cl contents in PBQS after oxidation are respectively, 47.42%, 1.23%, 22.60%, 23.0%, 0.62%, and 0.91%, indicating that the C/S, C/O, and C/H molar ratios are respectively, 5.5, 2.8, and 3.2, which are roughly consistent with the theoretical values of 6, 3, and 3  and C–OH (at 1239 or 1216 cm–1), are observed for both PBQS samples, indicating that hydroquinone units still exist even after oxidation. However, the disappearance of the broad peak of O–H (at 3129 cm–1) before oxidation, combined with the increase of relative intensity of C=O (at 1647 cm–1) after oxidation, verifies that the majority of hydroquinone units were converted into quinone units after oxidation by DDQ. Due to the existence of unoxidized hydroquinone, absorbed water is also inevitable, as deduced from the broad and strong peak at 3427 or 3434 cm–1.FTIR (Fourier‐transform infrared) spectra of PBQS samples were recorded to compare the structures before and after oxidation Figure1a. The2O. Thermogravimetric (TG) analysis was performed to estimate the content of absorbed water in PBQS  both refer to C–S–C(H) bond. The above XPS analysis agree well with the FTIR spectra, and based on all the characterization results, we can conclude that our final PBQS product is a polymer containing benzoquinone units in the majority and hydroquinone units in the minority linked by thioether bonds, with unavoidable residual NMP and absorbed H2O in it. Although this is not a perfect structure as we proposed in Scheme XPS (X‐ray photoelectron spectroscopy) analysis was also conducted for PBQS sample after oxidation Figure c–f. Gene2.2+/Li) and high theoretical capacity (496 mA h g−1). However, its serious volatility and solubility in organic solvents make it impossible to be directly applied in conventional Li battery with liquid electrolyte.Figurem LiTFSI/DOL + DME  electrolyte. Even with a lowered active material loading of only 30%, the cycling stability of BQ is still very poor that the capacity retention is below 32% after 20 cycles, in spite of the very high initial discharge capacity (429 mA h g−1). As expected, after polymerization, PBQS cathode with 60% loading exhibits dramatically improved cycling stability, with high reversible capacity of 275 mA h g−1. This result once again proves that polymerization through thioether bonds is a simple but effective approach to guarantee the cycling stability while not to reduce the capacity too much.BQ is one of the most attractive organic cathode materials due to its high redox potential  will gradually lower the oxidizing ability of remaining BQ units by electron‐donating effect, resulting in a sloping discharge curve.In Figure n structure units, namely (BQS)n , to simulate the structure of PBQS . Compared to the LUMO energy of BQ monomer (–3.54 eV), (BQS)n shows n lowered and nearly degenerate orbitals (energy level width = 0.09–0.12 eV), with descending LUMO energies of –3.74, –3.77, –3.79, and –3.80 eV for (BQS)3, (BQS)5, (BQS)7, and (BQS)9, respectively , suggesting that higher polymerization degree will slightly promote the starting reduction potential of PBQS. Considering both the representativeness of polymer structure and the complexity of calculation, we chose (BQS)5 as simulated PBQS for our further calculation. We were also concerned about polymers with residual hydroquinone groups [H2(BQS)5], and in different reduction states , in the structures of which O–H and O···Li bonds were symmetrically and homogeneously distributed in the chain . When we check the effect of residual hydroquinone units, it is surprising that the LUMO energy is lowered from –3.77 eV of (BQS)5 to –3.90 eV of H2(BQS)5 5 shows even much lower LUMO energy of –4.42 eV. This abnormal phenomenon for H2(BQS)5 and Li2(BQS)5 is probably due to the pseudo‐Jahn−Teller effect (PJTE),5 5 to –3.98 eV of Li4(BQS)5, –3.72 eV of Li6(BQS)5, and –3.49 eV of Li8(BQS)5. The negative LUMO energy shift from BQ to H2(BQS)5 (–0.36 eV), and positive LUMO energy shift from Li2(BQS)5 to Li8(BQS)5 (0.93 eV), can roughly agree with the starting reduction potential rise from BQ to PBQS (0.5 V), and the wide discharge potential range (1.3 V) of PBQS, respectively. Thus the different discharge behaviors of BQ and PBQS can be well illustrated by the simplified model we built, considering only the molecular geometric structure and ignoring many other factors such as intermolecular interaction, crystal structure, electrolyte, etc. We believe this calculation method is a simple but effective approach to predict the electrochemical behavior of such kind of polymer electrodes.The different electrochemical behaviors were also studied semiquantitatively by theoretical calculation. Since lowest unoccupied molecular orbital (LUMO) energy level is an efficient parameter to estimate the relative redox potential of an organic electrode material2.3m LiTFSI/DOL + DME electrolyte, we first concern about the practical reversible capacity of PBQS. Based on the two‐electron reaction of the perfect PBQS structure  coming from both benzoquinone and hydroquinone units [deducting O content in residual NMP (≈0.7%) and absorbed H2O (≈3.6%) from the total O content (22.6%) measured by elemental analysis], the theoretical capacity is reduced to 307 mA h g–1. At a current rate of 50 mA g–1, our PBQS product shows a reversible specific capacity of 275 mA h g−1 after oxidation, or only 165 mA h g−1 before oxidation  between the initial discharge process (232 mA h g–1) and the next charge process (272 mA h g–1), indicates the electrochemical oxidation of remaining hydroquinone groups, thus then a much higher discharge capacity of 260 mA h g–1 can be released in the second cycle. Similarly, the discharge capacity is gradually increased in the following cycles to reach a stable value (275 mA h g–1) at about the 10th cycle.For the comprehensive electrochemical performance of Li–PBQS battery with 1 on Figure3a, rea–1, the capacity retention is 98%, 94%, 90%, 86%, 80%, or 72%, at a current rate of 100, 200, 500, 1000, 2000 or 5000 mA g–1, respectively , releasing a large capacity of 198 mA h g–1 in less than 2.5 min. Such excellent high rate performance was rarely seen for either organic or inorganic electrode materials, especially considering that the electrode was fabricated without special techniques such as compositing with carbon nanotube–2) is not low for insulating organic materials. We ascribe the outstanding rate capability mainly to the fast redox kinetics of quinone groups,–1. After the initial capacity promotion (from 184 to 231 mA h g–1) and the following slow activation process, the reversible capacity reaches the highest value of 246 mA h g–1 at about the 140th cycle and then starts to slowly decrease with a decay rate of 0.04 mA h g–1 cycle–1, and thus the capacity still remains 86% at the 1000th cycle. We believe such cycling stability is much better than that of most reported organic electrode materials and can well satisfy the demand of practical use. It should be noted that, the Coulombic efficiency is only ≈95% at a low current rate of 50 mA g–1 . This side reaction may possess much slower reaction kinetics than benzoquinone units, so it can be inhibited at large current rate, leading to a high Coulombic efficiency. Based on above data and to our knowledge, PBQS represents the highest level in comprehensive battery performance of organic cathode materials, including not only high energy density, but also high rate capability and high cycling stability.Figure y Figure c. For th2.4+ and Na+. In this respect, organic electrode materials, especially those based on conjugated‐carbonyl, show their special superiority that Li‐free materials can be directly applied in rechargeable Na batteries,m NaTFSI/DOL + DME electrolyte with Na anode.Na‐ion battery is recognized as an ideal alternative to Li‐ion battery in recent years, in concern of the resource abundance of Li and Na element on the earth.Figure–1, both the initial and maximum discharge capacity  are approximate to those of Li–PBQS battery (232 and 275 mA h g–1), confirming that benzoquinone units in PBQS can be also fully utilized in spite of much large ionic radius of Na+. The discharge/charge curves are also similar to those of Li–PBQS battery, except the lowered average discharge voltage (2.08 V) as expected. Based on above data, PBQS can also achieve a high energy density of 557 W h kg−1 (2.08 V × 268 mA h g−1) in Na battery, which is superior to that of most inorganic cathodes for Na‐ion batteries.–1 cycle–1 and thus the capacity remains only 68% after 100 cycles. For the rate performance of Na–PBQS battery, it is found the charge capacity becomes larger and instable when the current rate is above 200 mA g−1 , which we ascribe to the inner short circuit caused by sodium dendrites formed in charge process at large current. We suspect there is probably some compatibility problem between Na anode and the 1 m NaTFSI/DOL + DME electrolyte, after all, this electrolyte is barely used in previously reported Na‐ion batteries. Therefore, we attempted to improve the electrochemical performance of Na–PBQS battery by adopting a more commonly used electrolyte for Na‐ion batteries, 1 m NaPF6/EC + DEC . Unfortunately, both the reversible capacity and cycling stability become even poorer, while 1 m LiPF6/EC + DEC electrolyte for Li–PBQS battery shows similar result , which together prove the incompatibility of such kind of electrolyte with PBQS cathode.2.5Figure–1. The similarity of CV curves verifies the same redox reaction mechanism, with either Li+ or Na+ as counterion. The two pairs of redox peaks can be assigned to the well‐known two‐step reaction of quinone unit (Q/Q•– and Q•–/Q2–), and the serious overlapping is mainly due to peak broadening effect of polymerization, which was observed in many other polymer electrode materials.+/Li and Na+/Na couple (0.33 V) by normalizing the potential axis, it is found the absolute redox potential of PBQS in Na battery still shifts to the negative side by about 0.3 V, which indicates larger ionic radius of Na+ brings about larger energy barrier to overcome in the reduction of PBQS.We were curious about the electrochemical behavior difference between Li–PBQS battery and Na–PBQS battery and investigated it by many electrochemical techniques Figure5. Figu–1, the electrochemical polarization of Li–PBQS system is very small in either discharge or charge process, benefitting from the high redox reversibility of quinone. However, the gap between average discharge and charge voltage is obviously increased from 0.17 V for Li battery to 0.45 V for Na battery. Besides the large overpotential between the actual discharge curve and the QOCV curve, an obvious voltage hysteresis is also observed that a negative or positive overpotential appears at the very beginning of each discharge or charge step after 2 h relaxation, respectively. We suspected this abnormal phenomenon is related to the stripping/deposition reaction of Na anode in 1 m NaTFSI/DOL + DME electrolyte, so we tested the discharge/charge behavior of Na electrode using a Na/Na/Na three‐electrode battery , the initial charge and next discharge process of Na electrode , show stable overpotentials of 0.22 and 0.06 V after the voltage hysteresis at the beginning. The voltage hysteresis is probably because of the large energy barrier for breaking the thick passivation layer on Na surface,+ with Na+ will only lower the absolute redox potential, but will not obviously affect the utilization and reversibility of PBQS electrode.Figure 32S as raw materials. As a polymer cathode for rechargeable Li battery, PBQS shows different discharge/charge behavior from BQ monomer, which is well illustrated by an energy level diagram obtained by DFT calculation. Benefiting from the high redox potential, high theoretical capacity and fast reaction kinetics of benzoquinone units, as well as intrinsic insolubility of polymer chains linked by thioether bonds, PBQS demonstrates superior comprehensive battery performance, including not only high energy density (2.67 V × 275 mA h g−1 = 734 W h kg−1) exceeding those of commercial inorganic cathodes  and other insoluble organic cathodes, but also stable long‐term cycling performance , as well as excellent fast‐discharge/charge ability . Due to the specialty of conjugated‐carbonyl, PBQS was also directly applied as cathode for rechargeable Na battery, showing similar discharge/charge behavior and gaining high energy density (2.08 V × 268 mA h g−1 = 557 W h kg−1) as well. The unsatisfactory cycling and rate performance of Na–PBQS battery are mainly ascribed to the poor compatibility between Na anode and the 1 m NaTFSI/DOL + DME electrolyte. Since the structure of our PBQS product is not perfect , there is still room to further improve the energy density to nearly 1000 W h kg−1 (in Li battery) by optimizing the synthesis conditions. We also look forward to better Na–PBQS battery performance by optimizing the electrolytes. We believe, our preliminary investigations on PBQS show the enormous possibilities of organic cathode materials, encouraging researchers to explore more and more high‐energy organic cathodes, toward the next generation of high‐performance, low‐cost, and sustainable energy storage devices.In summary, we developed a novel polymer cathode material by linking benzoquinone units with thioether bonds, namely PBQS, which can essentially solve the dissolution problem while largely maintain the high energy density of benzoquinone monomer. Although the chemical synthesis of benzoquinone polymer is difficult due to its high reactivity, PBQS is successfully synthesized with high yield through a polymerization–oxidation approach using DCBQ and LiAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The objective of this study was to apply extended NO analysis for measurements of NO dynamics in the lung, divided into alveolar and airway contribution, in amateur runners and marathoners.ENO) with estimation of alveolar NO (CANO) and airway flux (JawNO), ventilation, pulse oximetry, and peak flow were performed before, immediately after, and 1 hour after completing the race.The athletes participated in either a marathon or a half marathon. The athletes self-reported their age, weight, height, training distance per week, competing distance, cardio-pulmonary health, atopic status, and use of tobacco. Measurements of exhaled NO (Fp = 0.041), and marathoners, 3.6 ± 1.9 ppb (p = 0.002), than in control subjects, 1.4 ± 0.5 ppb. JawNO was higher in marathoners, 0.90 ± 0.02 nL s−1 (p = 0.044), compared with controls, 0.36 ± 0.02 nL s−1, whereas the increase in amateur runners, 0.56 ± 0.02 nL s−1, did not attain statistical significance (p = 0.165). Immediately after the race there was a decrease in FENO in both amateur runners and marathoners, whereas CANO and JawNO were decreased in marathoners only.At baseline the alveolar NO was higher in amateur runners, 2.9 ± 1.1 ppb (Our results support the view that there is an adaptation of the lung to exercise. Thus strenuous exercise increased both airway and alveolar NO, and this might in turn facilitate oxygen uptake. Nitric oxide (NO) is a gaseous signalling molecule with a variety of biological actions in the human body. It is an important mediator of homeostasis by regulating vascular tone, platelet aggregation, bronchomotor tone, and neurotransmission . FurtherDuring prolonged, repetitive, and intense physical activity the body undergoes adaptations to meet the increased metabolic demand. The appropriate cardiovascular and respiratory responses to exercise are fundamental for adequate oxygen supply. The muscle, cardiovascular, and hematologic systems adapt both structurally and functionally to increase the maximal aerobic capacity . It is wENO) has been widely investigated, but there has been no consensus. This is unsurprising as there are significant differences in methodology between the studies. The majority of studies demonstrate decreased FENO after exercise, but a minority demonstrate increased FENO with focus on cardiovascular effects  referred to as amateur runners, and above 50 km (n = 10) referred to as marathoners. The study protocol was explained to the subjects. The study subjects self-reported their age, weight, height, training distance per week, competing distance, cardio-pulmonary health, atopic status, and use of tobacco. The subjects were examined and followed up in a location close to the finish line at the Department of Physiology III, Karolinska Institute, Stockholm, Sweden. The same methodology of NO analysis has been applied in a previous study (n = 10) from that study were used as controls.Twenty participants in a half marathon or a full marathon race were enrolled. They were divided into two groups based on their weekly running distance: under 50 km . Validations of the calibration and flow rate of the sampling system were made on a daily basis, and the zero was ensured before each measurement. The expiratory pressure for all subjects ranged between 5 cm H2O and 20 cm H2O in order to exclude a NO contribution from the nasal cavity. Exhaled NO from the mouth was measured at three flows above 0.1 L s−1  and abbreviated FENO0.1 for 0.1 L s−1. Exhalation from the nose was measured at 0.1 L s−1 (FnNO0.1). The mean values of three breaths  of each flow rate were used for statistical analysis.NO measurements were performed in accordance with the American Thoracic Society/European Respiratory Society (ATS/ERS) recommendations  but with−1, and mean values of two exhalations at each flow were used  and airway flux of NO values  from a plot of flow rate and exhaled NO production from the respiratory system.The linear model of Tsoukias and George  was applere used . The val2), peripheral oxygen saturation (SpO2), end-tidal CO2 (ETCO2), respiratory rate (resp rate), and heart rate (HR). A Mini-Wright standard peak flow meter  was used to identify airway obstruction after running.A CS/3 critical care monitor  was used to measure minute ventilation, oxygen consumption . Correlations were tested with Spearman rank order correlation. Differences between the groups were tested with Kruskal–Wallis test and Mann–Whitney U test. Differences within the groups were tested with Friedman ANOVA and Wilcoxon signed rank test. SPSS v. 21 for Windows  was used for all statistical calculations. Data have been presented as arithmetic mean and quartiles and as geometrical mean and quartiles for FENO0.1 and JawNO. A p value of <0.05 was regarded as significant.The normal distribution of the data was tested with the Kolmogorov–Smirnov test. NO parameters that could not be proven to have a normal distribution were treated as non-parametric  for control subjects, 0.97  for amateur runners, and 0.97  for marathoners.ENO0.1 was higher in runners (p = 0.015) and marathoners (p < 0.001) compared with controls, but JawNO was higher in marathoners only (p = 0.04). There was an increase in CANO when comparing controls and amateur runners and controls and marathoners . In amateur runners there was a correlation between JawNO and training distance per week, r = .78, p = 0.007. In marathoners this correlation was negative, r = –.57, and failed to reach statistical significance (p = 0.087). A quadratic regression line was fitted to all athletes, and the adjusted 2r was .28. The p value for the regression coefficient was 0.008 for the linear term of the model and .013 for the quadratic term  km, as compared with those of amateur runners, 26  km (tic term .p = 0.005) and a decrease in FENO0.1 (p = 0.022) in comparison with before the race. One hour later the HR was still increased (p = 0.005). In marathoners the HR was increased (p = 0.005), and ETCO2 (p = 0.005) and FENO0.1 (p = 0.005) decreased immediately after the race and 1 hour later . JawNO (p = 0.022) and CANO (p = 0.037) decreased immediately after the race in marathoners . In amateur runners there was a decrease in nasal NO immediately (p = 0.011) and 1 hour after the race (p = 0.028). The race had no impact on nasal NO in marathoners , and respiratory rate were measured only in the four runners and six marathoners who participated in the half marathon (p = 0.017), and 1 hour after the race (p = 0.007), while oxygen uptake was only increased immediately after the race (p = 0.019). Respiratory rates were decreased 1 hour after the race (p = 0.016).Minute ventilation, oxygen consumption  after their race. Based on these results, we hypothesized that the oedema could be formed due to a decrease in alveolar NO, but we did not assess oedema formation.In athletes performing strenuous exercise there is a risk of hypoxaemia and lung oedema, and highly trained athletes are at risk to develop hypoxaemia due to heavy exercise . When ththe lung . Thus ththe lung . In our 2-max) as well. One limiting factor for VO2-max is the pulmonary diffusing capacity, which is more evident in elite-trained athletes due to an increased cardiac output with a subsequent decrease in red blood cell transit time. Increased surface area of gas exchange could possibly decrease this limiting factor. This could be done by either angiogenesis of the pulmonary system and/or increased CANO with dilation of the pulmonary capillary bed and subsequent increase in ventilation/perfusion matching. Our data concerning the correlation of ETCO2 to CANO might suggest that NO is involved in oxygen diffusion and ventilation.Endurance-trained athletes have an increased maximum oxygen uptake (VOThe role of NO in the airway epithelia has been extensively investigated. NO is important in the defence system, since it has anti-microbial, anti-inflammatory, and anti-oxidative properties, and also by regulating the ciliary function of the respiratory epithelium that mechanically transports inhaled particles . In our Intense and prolonged exercise is associated with increased risk for upper respiratory tract infection due to alterations in the immune system . NO is kWe conclude that our finding of increased alveolar NO in amateur runners and marathoners suggests that there is a potential for the lung to adapt to strenuous exercise. Such a capacity change of the lung might be accompanied by an enhanced oxygen uptake due to the vascular effects of NO that in turn improve the perfusion of the alveoli. This hypothesis needs further investigation in other endurance athletes such as elite bicycle riders and skiers.


Venous blood gas (VBG) is frequently used in the neonatal unit as alternative for capillary blood gas (CBG). However, studies reporting correlation are conflicting and data on interchangeability in neonates are lacking.We investigated the correlation and interchangeability of the components between VBG and CBG in infants admitted to the neonatal intensive care unit.2) (±0.67 kPa = 5 mmHg); partial pressure of oxygen (pO2) (±0.67 kPa = 5 mmHg); base excess ± 3 mmol/l; and bicarbonate (HCO3−) ± 3 mmol/l.In a prospective study in the neonatal unit in Leiden University Medical Center (Netherlands), simultaneously VBG and CBG were withdrawn in neonates when both venous puncture and intravenous access as blood gas monitoring was indicated. From each blood gas analysis, a Pearson correlation, intraclass correlation, and Bland–Altman analysis was performed. Clinically acceptable difference for each blood gas value was defined up-front by means of an absolute difference: pH ± 0.05; partial pressure of carbon dioxide , BE  and bicarbonate ; strong for pCO2 ; and moderate for pO2 . The percentage of the interchangeability within our acceptable absolute difference for pH was 88%, pCO2 72%, pO2 55%, BE 90%, and bicarbonate 94%.In 93 patients [median gestational age 31 (IQR 29–34) weeks], 193 paired samples of VBG and CBG were collected. The Pearson correlation between VBG and CBG was very strong for pH (2.VBG and CBG in neonates are well correlated and mostly interchangeable, except for pO Patients admitted to the neonatal intensive care unit (NICU) frequently need monitoring of gas exchange and oxygenation . Blood g3−) between CBG and VBG, but the results in pCO2 were conflicting  as an alternative for CBG , as venoflicting –9, 15. Hflicting .To test the hypothesis that VBG is a good substitute for CBG monitoring for metabolic state, oxygenation, and gas exchange, we determined the correlation and interchangeability between VBG and CBG in neonates admitted to our NICU.g a One-step safety lance green (1.8 mm)/BD microtainer Quickheel lancet (0.8 mm depth), and for preterm >1,500 g a One-step safety lance blue (2.3 mm)/BD microtainer Quickheel lancet (1 mm depth). The heel lances are only performed at the lateral or medial part of the heel, never at the center, conform the suggestions of Noureldein et al. . All patients needing blood gas analysis, were eligible and parents were asked for informed consent by a member of the medical staff. No incentives were offered , pO2 (± 0.66 kPa = 5 mmHg), BE (±3 mmol/l/), and bicarbonate (±3 mmol/l), based on bias of reference values of infant’s blood gas (A Pearson correlation and intraclass coefficient (two-way mixed and absolute agreement) of the different parts of the blood gases were calculated. To determine interchangeability, a Bland–Altman analysis was performed, based on the 95% limits of agreement (LoA), estimated by mean difference (bias) ±2 SD of the differences, that provides a 95% confidence interval of the differences between VBG and CBG are expected to lie. Clinically acceptable difference for each blood gas value was defined up-front by means of an absolute difference: pH (±0.05), pCOlood gas , 20 and lood gas . The oxy2   kPa] . In ordeIn total, 193 paired samples of VBG and CBG of 93 neonates were analyzed (patient characteristics: Table r = 0.79), BE (r = 0.90), and bicarbonate (r = 0.87), strong positively correlated for pCO2 (r = 0.68) and weak positively correlated for pO2 (r = 0.31) (Table 2 (ICC = 0.802); BE (ICC = 0.946), bicarbonate (ICC = 0.928) and fair agreement for pO2 (ICC = 0.364) , and pO2 .The VBG and CBG was significant and very strong positively correlated for pH  for most intensive care pediatric and neonatal patients but not when there was congestive heart failure or shock  was lower in our study when compared to the 90% in pediatric and adults studies  could have occurred, as this has more influence on the pCO2 than on the metabolic component. Tachypnea due to pain and discomfort during venepuncture could also have influenced the pCO2, as a large proportion of our cohort was not sedated, while most children and adults in the other studies were probably sedated. Sampling from a venous central line (2 outside the clinical acceptable difference. It is also possible that the puncture was more difficult in a crying and uncomfortable neonate, and increased the chance for technical errors. For analysis, we assumed that all samples per individual were independent separate measurements as they were taken spread in time at three different moments during the NICU admission and not performed subsequently after each other (We reported a high degree of interchangeability for the different components of blood gas. The venous pH, BE, and bicarbonate were for more than 88% interchangeable and therefore a reliable substitute for CBG, similar to previous results of pediatric and adults studies , 22, 23. studies , 17, 23.ral line , 22, 23 ch other .2 and not for pO2 can be explained by the oxygen–hemoglobin dissociation curve and carbon oxide equilibrium curve. A previous study in adults reported that pO2 differ more in arterial, capillary and venous values than pCO2 (2 more in venous blood compared to arterial or arterialized capillary blood. In contrast to pO2, pCO2 is described in a linear carbon dioxide equilibrium curve for both arterial as venous blood. The carrying capacity of blood CO2 is much greater than for O2, and blood can load and unload large amounts of CO2 with small difference in gas tension, for the regulation of gas exchange and acid–base balance (2 values between arterialized capillary and venous are comparable, and the pO2 not.The good correlation of pCOhan pCO2 . This ca balance . TherefoOur findings support the use of VBG in neonates for metabolic evaluation and monitoring gas exchange, but not for monitoring oxygenation. In most occasions, the blood gasses are interchangeable; therefore when a venepuncture is performed, a VBG can be withdrawn simultaneously to limit the burden for patient.Medical ethical committee of Leiden Medical University approved this study, including the recruiting method and asking parents or caregivers for consent by trained caregivers. All the participants from whom we collected the data, a written consent is available.RT carried out the data collection, data analyses, and initially wrote, revised, reviewed, and approved the manuscript. AP conceptualized and designed the study, and reviewed, revised, and approved the manuscript. SP critically reviewed the data analyses, and reviewed and approved the final manuscript. EL, VS, and EL critically reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to establish a baseline titre for the population of Mumbai, Maharashtra, India. Four hundred healthy blood donors, attending blood donation camps, were screened using a survey questionnaire. Widal tube agglutination test was performed on the diluted sera  of blood donors, with final dilution ranging from 1 : 40 to 1 : 320. Salmonella enterica serotype Typhi were found to be 1 : 40 and 1 : 80, respectively. Similarly, the baseline antibody titres for the H antigen of Salmonella enterica serotypes Paratyphi A and Paratyphi B were found to be 1 : 40 and 1 : 80, respectively. Out of 400 individuals providing samples, 78 (19.5%) individuals showed antibody titres ≥ 1 : 40 for at least one antigen and 322 (80.5%) showed no agglutination. The baseline antibody titres against O antigen and H antigen of S. Typhi O antigen and titre of ≥ 1 : 160 for both S. Typhi H antigen and S. Paratyphi BH antigen. Antibody titre of ≥ 1 : 80 can be considered significant for S. Paratyphi AH antigen. Thus, it was noted that the diagnostically significant cutoff of antibody titre from acute phase sample was ≥ 1 : 80 for Salmonella enterica subspecies enterica serotype Typhi and paratyphoid fever caused by Salmonella enterica subspecies enterica serotypes Paratyphi A, Paratyphi B, and Paratyphi C [ Salmonella enterica in the blood. However, in healthy populations of the endemic areas, the antibody titre may be found to be high in the presence of cross-reacting antigens owing to other infections, a healthy carrier state, long-standing liver disease, and previous typhoid vaccination [ Salmonella infection or history of vaccination against Salmonella [The term enteric fever includes typhoid fever, caused byatyphi C . Endemicatyphi C . The criatyphi C . Among tcination . Therefocination . Howevercination –8. Furthlmonella . In ordelmonella . Substanlmonella –13. The N = 400), of both genders and those aged 18 to 55 years, who attended the various blood donation camps organized by the institution during the period extending from May 2014 to September 2014.This study was a cross-sectional type of study carried out at the Department of Microbiology, Grant Government Medical College and Sir J. J. Group of Hospitals, Mumbai, Maharashtra, India, after obtaining an approval from the Institutional Ethics Committee. Blood samples were collected after obtaining written informed consent from healthy blood donors  vaccine or oral typhoid vaccine/Vi vaccine nor had suffered from enteric fever in the past. We selected only those blood donors who were permanent residents of Mumbai, India.Individuals with any active or recent infections including hepatitis B, hepatitis C, malaria, Dengue fever, brucellosis, and HIV/AIDS and any type of fever in the last 6 months were excluded from this study. Salmonella antigen test kit for tube test was provided by Star Diagnostics Pvt. Ltd., India. This test kit contained smooth suspensions of antigens “O” and “H” of Salmonella serotype Typhi, “H” antigen of Salmonella serotype Paratyphi A, and “H” antigen of Salmonella serotype Paratyphi B.A venous blood sample of 5 ml was collected in a plain tube and was allowed to clot at room temperature for about 30 minutes. The samples were then centrifuged at 3000 rpm (revolutions per minute) for 10 minutes in order to separate the serum from the blood. Sera were transferred to clean and dry sterile storage vials. The serum samples were refrigerated at −20°C. StainedThe test was performed after bringing sera and reagents to room temperature. Widal titre was estimated by confirmatory quantitative tube agglutination test using standard agglutination test procedure . As per The method used for finding the baseline titre was Widal tube agglutination test. The amount of antigen suspension used was the same as the amount of diluted sera making the dilution 2-fold, and hence the readings in our study begin with titre 1 : 40, which is in accordance with the manufacturer's instructions and also the standard lab protocol followed by the Department of Microbiology of the institution where our study was conducted.Data was analyzed in an Excel sheet and the variables were summarized using frequency count and percentage. Salmonella enterica, using Widal tube agglutination test. Blood donors were in the age group of 18 to 55 years. Out of all participant blood donors, 352 (88%) were males. Out of the 400 individuals, 78 individuals (19.5%) were found to be positive (≥1 : 40) for at least one of the antigens. 322 individuals (80.5%) were found to be negative (<1 : 40) for all antigens  were found positive (≥1 : 40) for S. Typhi “H” antigen. Only 2 (0.5%) individuals were positive (≥1 : 40) for S. Paratyphi AH antigen. The total number of individuals positive (≥1 : 40) for S. Paratyphi BH antigen was 15 (3.75%).As seen in  S. Typhi, 20 (5%) individuals had a titre of 1 : 40, 14 (3.5%) individuals had a titre of 1 : 80, 1 (0.25%) individual had a titre of 1 : 160, and 2 (0.5%) individuals had a titre of 1 : 320. Similarly, among the total of 41 (10.25%) individuals positive against H antigen of S. Typhi, 11 (2.75%) individuals had a titre of 1 : 40, 25 (6.25%) individuals had a titre of 1 : 80, and 5 (1.25%) individuals had a higher titre of 1 : 160.As seen in  S. Paratyphi A, 1 (0.25%) individual had a titre of 1 : 40 and the other individual had a titre of 1 : 80. It can be seen that very few individuals (0.5%) were positive for H antigen of S. Paratyphi A.As seen in  S. Paratyphi B, 4 (1%) individuals had a titre of 1 : 40, 7 (1.75%) had a titre of 1 : 80, and only 4 (1%) samples reported a higher titre of 1 : 160 (Among 15 (3.75%) individuals positive against H antigen of 1 : 160 . Salmonella from blood or bone marrow has always been the gold standard for diagnosis of enteric fever. However, most healthcare centers in India do not have easy access to this method and also culture methods are seen as time-consuming. The Widal agglutination test has always been the preferred test for diagnosis of enteric fever in many developing countries, including India, where it is extensively practiced. Especially in endemic areas, the Widal test is still of significant diagnostic value, provided judicious interpretation of the test is made against a background of pertinent information, especially data which relate to agglutinin levels in normal individuals and in nontyphoidal fevers common in the region [Isolation ofe region . Salmonella enterica. Our study has shown that disparate titres of antibody, against the different serotypes of Salmonella enterica, exist in healthy individuals.This study was the first ever study done in Mumbai, to the best of our knowledge, which was conducted for the estimation of baseline antibody titre in healthy individuals against serotypes Typhi and Paratyphi A and Paratyphi B of S. Typhi H antigen. Taking these results into consideration, the baseline antibody titres against O antigen and H antigen of Salmonella enterica serotype Typhi were found to be 1 : 40 and 1 : 80, respectively. Our results are in concordance with those reported in studies conducted by researchers in other states of India that the disease is endemic to, such as Karnataka and Uttarakhand, where the recorded baseline titres against O antigen and H antigen of S. Typhi were found to be 1 : 40 and 1 : 80, respectively, in both states [ Salmonella enterica serotype Typhi, for a possible diagnosis of typhoid fever for Kathmandu Valley, Nepal [The results of our study established the presence of high antibody titres in healthy individuals. Out of 78 (19.5%) healthy individuals with positive titres for at least one antigen, we found that 37 (9.25%) had anti-O antibody titres of ≥ 1 : 40 and 41 10.25%) had antibody titres of ≥ 1 : 80 against.25% had h states . A simily, Nepal . S. Typhi were 1 : 320 and 1 : 160 in 0.5% and 1.25%, respectively. Such higher titres of 1 : 160 against O and H antigens of S. Typhi were seen in a study carried out in Kerala, India, by Sreenath [ S. Paratyphi A. This study demonstrated that anti-H agglutinin titre to Salmonella enterica serotypes Paratyphi A and Paratyphi B in Mumbai was lower than that against Salmonella enterica serotype Typhi. A total of 15 individuals had anti-H titre ≥ 1 : 40 to Salmonella enterica serotype Paratyphi B. A very little proportion of individuals showed significant levels of anti-H titre against Salmonella enterica serotype Paratyphi A, indicating that it is not a usual serotype in Mumbai.In this study, the greatest anti-O and anti-H titres againstSreenath . We obta Salmonella antigen, infection with other Enterobacteriaceae, and other diseases such as malaria and Dengue fever and also due to variability and poor standardization of commercial antigen preparation. False positive agglutination can also be seen as a cross reaction with nontyphoidal Salmonella [ Salmonella Typhi, and the ability to select an appropriate diagnostic cutoff titre [Widal agglutination test can be positive in individuals with previous immunization againstlmonella . A studyff titre . Though Various studies which have been carried out in different states of India have shown that the baseline titre is different in different geographical locations. Hence, the baseline titre of a specified geographical area should first be established –12. Typh Salmonella enterica serotype Typhi were found to be 1 : 40 and 1 : 80, respectively. Similarly, the baseline antibody titres for the H antigen of Salmonella enterica serotype Paratyphi A and Salmonella enterica serotype Paratyphi B were found to be 1 : 40 and 1 : 80, respectively. Thus, an antibody titre, from acute phase sample, of ≥ 1 : 80 for S. Typhi O antigen and titre of ≥ 1 : 160 for both S. Typhi H and S. Paratyphi B H antigens and antibody titre of ≥ 1 : 80 for H antigen of S. Paratyphi A can be considered as diagnostically significant.Widal test results from an acute phase serum sample should be interpreted based on the baseline titre prevalent in healthy individuals in that particular geographic area. In this study, the baseline antibody titres against O antigen and H antigen ofThe participant blood donors were surveyed using a questionnaire. Only those who had never been vaccinated with TAB or oral typhoid vaccine and who had not suffered from enteric fever in the past, were selected for the study. Participating blood donors were informed and explained about the study and consent was obtained.