Our study aimed to investigate the ocular surface health of Shanghai University students.This is a cross-sectional study carried out among freshmen and sophomores on the main campus of Shanghai University. Questionnaires including the widely-used ocular surface disease index (OSDI) and the Zung Self-rating Anxiety Scale (SAS) were completed first, and then ocular examinations were conducted regarding height & weight, blood pressure and heart rate, optometry, intraocular pressure exam, vision and subjective refraction, Aladdin, Macular pigment density measurement, tear test, anterior segment examination, fundus photography, ophthalmologist check, TOPCON OCT check, and Collin’s fundus blood test.n = 40) had moderate or severe anxiety, 78% were mild and a small portion (17.5%) didn’t have anxiety at all. No statistical significant association was found between anxiety with DED, fluorescein staining or with corneal sensation . However, subjects with DED had more symptoms of anxiety. Results also showed that students who kept eye strain for a long time were more inclined to have DED ; those who watched mobile phones and/or computers for over eight hours daily were more vulnerable to DED and fluorescein staining than others .Totally 901 students were involved in our five-day study. The prevalence of myopia was 92%  < − 0.50 D), and that of high myopia was 23% (SER < − 6.0D). The prevalence of dry eye disease (DED) was 10%. The corneal epithelial loss rate  was 10%, and corneal sensation decline rate (≤ 30 mm) was 12%. 4.5% of subjects  contains supplementary material, which is available to authorized users. Eye health, including ocular surface health, is an integral part of the human physical health. Statistics from the World Health Organization (WHO) alarms us against the severity of eye health. A total of nearly 50 million persons are blind and another 150 million are victims of severe visual disability, and this number will probably double in the year 2020 [China, with its 1.5 billion population, has a huge number of students. As reported in the 2016 National Statistical Communiqué on Development of Education published by the Ministry of Education, there were about 160 million students from grade 1 to 12, and over 41 million college/university students (including nearly 2 million post-graduate students) at school at the survey time .According to the 2004 National Students Physical Health Monitoring Report, the poor sight detection rate was 32.5% among pupils aged 7 to 12. The rate was 59.4% among junior middle school students and 77.3% among senior middle school students. A larger proportion (80.0%) of university students were detected with poor sight and some of them even had high myopia .With the popularity of computers and smart phones/video display terminals (VDT), more and more Chinese students suffer from refractive errors and have to wear glasses if the error is not detected and corrected in a timely manner . While wDry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment, potentially accompanied by ocular surface damage. In 2016, the Asia Cornea Society (ADES) and the Dry Eye Society Japan implemented new diagnostic criteria for DED that enabled diagnosis with two positive items, namely subjective symptoms and decreased TBUT (≤ 5 s) .To date, teenagers-based studies in China have been focusing on myopia/high myopia, good examples of which may be the Shunyi Study , 10, theThe obvious strength of the updated study is that it is the first to pay attention to ocular surface health condition of Chinese university students, as well as to explore the association of their anxiety status with their eye health.This is an observational cross-sectional study, comprising a questionnaire survey and ophthalmologic examinations. It was carried out on Baoshan Campus of Shanghai University in September 2016. Located in Shanghai with three campuses, Shanghai University is one of the national key universities with over 54,000 students from various areas across the country to study in the university by February 27, 2017 . BaoshanP values were two-sided and were considered statistically significant when the P values were < 0.05. Person Chi-Square test was used to analyze associations with genders and ages, different eye-using habits and anxiety status. Cochran-Armitage Trend Test was performed to identify the trend of association between anxiety and DED.Statistical analysis was performed with SAS® software, version 9.2 . Spherical equivalent refraction (SER) was calculated as the spherical value of the refractive error plus half of the cylindrical value. Myopia was defined as a SER ≤ 0.5 diopters (D) and high myopia was defined as a SER ≤ 6.0 D. The OSD was determined based on the OSDI scores and the ophthalmologic examination results. Corneal epithelial loss was identified when the corneal fluorescein staining > 1. Corneal sensation ≤ 30 mm was considered as decline. Statistical analysis focused on OSDs, specifically the dry eye disease (DED), cornea fluorescein staining and corneal sensation. All Our study involved 901 students in total in Shanghai University. The subjects, at an equal sex ratio, responded with questionnaires and ophthalmologic examinations. Most of the subjects were freshmen and sophomores whose ages ranged from 18 to 22  < − 0.50 D), and that of high myopia was 23% (SER < − 6.0D). The prevalence of DED was 10%. The corneal epithelial loss rate  was 10%, and corneal sensation decline rate (≤ 30 mm) was 12%.n = 40, see Table p > 0.05). However, subjects with DED had more symptoms of anxiety (see Table p = 0.0023), getting upset easily or feeling panicky (p = 0.0004), and being bothered by headaches neck and back pain (p = 0.0101), feeling weak and getting tired easily (p = 0.0004), being bothered by dizzy spells (p = 0.0004), having fainting spells or feeling like it (p = 0.0175), being bothered by a stomach aches or indigestion (p = 0.0001), face getting hot and blushes (p = 0.0039). Cochran-Armitage Trend Test identified a trend of anxiety among DED subjects , feeling weak and getting tired easily (p = 0.0340), being bothered by dizzy spells (p = 0.0120), and being bothered by a stomach aches or indigestion (p = 0. 0190).Statistical analysis indicated that 4.5% of subjects ; those who watched mobile phones and/or computers for over eight hours daily were more vulnerable to DED and fluorescein staining than others . No significant differences were found among other variables such as age, gender, near work and improper reading gestures; no significant differences/associations for corneal sensation (see Table p < 0.05) after adjustment.Results also showed that students who kept eye strain for a long time were more inclined to have DED , that of high myopia was 23% (SER < − 6.0D), and dry eye disease 10%. The corneal epithelial loss rate  was 10%, and the corneal sensation decline rate (≤ 30 mm) was 12%.P > 0.05), which may be due to the shorter and less frequent use of contact lenses by these freshmen.There are quite a few published studies on myopia and ocular surface health of Chinese university students. The study implemented by Jing Sun et al. in Shanghai Donghua University found that 95.5% of the subjects were myopic (SER < − 0.50 D), 19.5% were highly myopic (SER < − 6.0 D) . This isp = 0.003; p < 0.001, respectively) [p > 0.05). A reasonable explanation may be that the subjects included in our study were young people (mean age 19.7 ± 2.7) with relatively good physical and mental health . Results of a study by van der Vaart et al. also found associations between DED and anxiety differed across age groups with the elders having stronger associations [Mental disorder, specifically anxiety, was explored in our study. As currently known, these factors are related to anxiety: Biological factors, such as genetic and physiological diseases, social factors, tension, and high work pressure, psychological factors, hormone levels, and so on. Published results of studies have already found correlation of eye diseases, especially the DED, with mental disorders. A study by Li M et al. in 2011 showed that the prevalence of anxiety or depression symptoms in dry eye syndrome subjects was significantly higher than in the control group (ctively) . A meta-ctively) . InvestiNonetheless, subjects with DED in our study were found to have more symptoms of anxiety, e.g. feeling afraid for no reason at all, getting upset easily or feeling panicky, feeling weak and getting tired easily, etc. ; and (2) watching mobile phones and/or computers for over 8 h every day, associated with the DED and corneal fluorescein staining . Near work, though considered as one of the reasons for refraction error [p > 0.05). One possible explanation may be that the variable of near work is a vague expression without a specific standard for subjects to make better or precise judgments. Another reason may be near work itself doesn’t do much harm to ocular surface disease, but it will do if combined with a long time and/or continuous use of VDT, a good example of which is the variable “watching mobile phones and/or computers for over 8 hours every day”. Our study identified the importance of period of time, either for eye strain or watching VDTs. This is consistent with findings of previous studies [Regarding habits, statistical significance was found in two habits out of ten  and the Zung Self-rating Anxiety Scale (SAS). (DOCX 19 kb)


Further testing with the MultiFlow® DNA Damage Assay revealed that TK6 cells exposed to NTP BCE, as well as a BCE reference material (BC XRM), exhibited a signature consistent with aneugenic activity in TK6 cells. Results from experiments reported herein confirmed these in vitro observations with NTP BCE and BC XRM. We extended these studies to include a novel test system, the MultiFlow Aneugen Molecular Mechanism Assay. For these experiments, TK6 cells were exposed to NTP BCE and BC XRM over a range of concentrations in the presence of fluorescent Taxol (488 Taxol). After 4 h, nuclei from lysed cells were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho‐histone H3 (p‐H3) and Ki‐67. Whereas BCEs did not affect p‐H3:Ki‐67 ratios (a signature of aneugenic mitotic kinase inhibitors), 488 Taxol‐associated fluorescence (a tubulin binder‐sensitive endpoint) was affected. More specifically, 488 Taxol‐associated fluorescence was reduced over the same concentration range that was previously observed to induce MN. These results provide direct evidence that BCEs destabilize microtubules in vitro, and this is the molecular mechanism responsible for the aneugenicity findings. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.Black cohosh extract (BCE) is a popular botanical dietary supplement marketed to relieve symptoms of various gynecological ailments. Studies conducted by the National Toxicology Program (NTP) showed that BCE induces micronucleated erythrocytes in female rats and mice. Subsequently, the NTP showed that a variety of BCEs, including the sample that induced micronuclei (MN)  Actaea racemosa) and is one of the top 10 selling botanical dietary supplements in the United States  is evaluating a number of botanicals for toxicological effects , and reference material root powders from red, yellow, and Chinese cohoshes in the In Vitro MicroFlow® assay, which uses flow cytometry to quantify micronuclei (MN) in mammalian cell cultures  . Whereas a large proportion of clastogens are DNA‐reactive, aneugenic agents are recognized as non‐DNA reactive, most often causing genotoxicity through tubulin binding and thereby disruption of mitotic spindle function, or via inhibition of mitotic kinase(s)  algorithms to evaluate changes in several biomarkers after 4 and 24 h of exposure. The biomarker responses include phosphorylation of H2AX (γH2AX), translocation of p53 to the nucleus, phosphorylation of histone H3 at serine 10 (p‐H3), and induction of polyploidy. Together with information on cytotoxicity at 24 h, the machine‐learning algorithms translate the collection of MultiFlow responses into predictions about predominant MoA—clastogenic, aneugenic, or non‐genotoxic  Assay , carbendazim (CAS no. 2068‐78‐2), and methyl methanesulfonate  were purchased from Millipore Sigma, St. Louis, MO. AMG‐900 (CAS no. 877399‐52‐5) and epothilone A (CAS no. 152044‐53‐6) were from Selleckchem, Houston, TX. NTP BCE (lot number 3012872) was procured from PlusPharma  and BC XRM (lot number ASB‐00030148‐005) was procured from ChromaDex . These samples of NTP BCE and BC XRM are the same materials that were tested by Smith‐Roe et al. , methotrexate , mebendazole , carbonyl cyanide e et al. .2 at or below 1 x 106 cells/mL. The culture medium consisted of RPMI 1640 with 200 μg/mL sodium pyruvate , 200 μM L‐glutamine, 50 units/mL penicillin and 50 μg/mL streptomycin , and 10% v/v heat‐inactivated horse serum .TK6 cells were purchased from ATCC® (cat. no. CRL‐8015) and grown in a humidified atmosphere at 37°C with 5% CO5/mL). NTP BCE and BC XRM were prepared in DMSO as a series of 100× stock solutions. Exposure occurred by delivering 2 μL stock solution per well (duplicate wells), for final BCE concentrations of 0, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 μg/mL. Concurrent positive controls were MEB (0.25 μM) and MTX (0.01 μM). Upon addition of test chemicals, the plates were immediately incubated in a humidified atmosphere at 37°C with 5% CO2 for 24 h. After the exposure period, cells were processed for MN frequencies via flow cytometric analysis using In Vitro MicroFlow® Kit reagents . These methods have been reported in detail elsewhere  containing 198 μL TK6 cell suspension . Stock photomultiplier tube detectors and associated optical filter sets were used to detect fluorescence emissions associated with the MicroFlow kit‐provided fluorochromes: SYTOX Green® (detected in the FITC channel) and ethidium monoazide . The stop mode for these analyses was the acquisition of 5000 EMA‐negative nuclei per replicate well.The NTP BCE and BC XRM micronucleus experiments described above were performed as two independent repeat experiments. Note that the same series of 100× stock solutions was used for both experiments.p < 0.05 was used to indicate a significant increase over concurrent solvent control . Note that %MN were log10 transformed to satisfy homogeneity of variance assumptions , and data from the two independent experiments were pooled for these analyses.Micronucleus data analyses were restricted to those concentrations that resulted in ≤55% reduction to relative nuclei counts and did not exceed 4× increase in EMA‐positive events, whichever was lowest. Using these criteria, we were able to analyze the highest concentrations tested, that is, 500 μg/mL. The effect of treatment on mean MN frequency was evaluated with a one‐sided Dunnett's test, and https://proastweb.rivm.nl; accessed June 27, 2019).Benchmark dose (BMD) analyses  were performed with pooled NTP BCE and BC XRM MN data. The critical effect size was set to 1 . “Compound” was selected as the covariate, and the resulting 95% confidence intervals (CIs) were used to represent the relative potency of the compounds. These methods were the same as those described previously  plus 2 μL of DMSO‐solubilized test chemical per well. Twenty concentrations were tested using a square root 2 dilution scheme—that is, each concentration differed from the one above by a factor of 70.71%. Both BCEs were tested up to 1000 μg/mL. Each concentration was tested in a single well, whereas solvent was evaluated in four replicate wells. Concurrent positive controls were MMS and carbendazim. Upon addition of test chemicals, the plates were immediately incubated in a humidified atmosphere at 37°C with 5% CO2 for 24 h.Chemicals were tested in U‐bottom 96‐well plates  containing 198 μL TK6 cell suspension , PE (B2 channel), propidium iodide (B3 channel), and Alexa Fluor® 647 (R1 channel). Flow cytometry data were analyzed using FlowJo software v10.5.0 .n and greater DNA‐associated propidium iodide fluorescence). Nuclei counts were used to derive Relative Nuclei Counts (RNC), and %cytotoxicity was calculated as 100% minus %RNC at 24 h.Representative bivariate graphs, gating logic, and position of regions were described in detail in earlier reports  predictions were made in two manners: comparing fold‐increase values against a global evaluation factor (GEF) rubric, and via an ML ensemble . Compound was selected as covariate, and the resulting 95% CIs were used to represent the relative potency of the compounds. As with the MN data, these analyses were performed with RIVM PROAST Web software v65.2.Unsupervised clustering was performed for test articles predicted to have aneugenic activity by the MultiFlow DNA Damage Assay. As described previously, aneugens group broadly into two subclasses: tubulin binders vs. mitotic kinase inhibitors . Suspected aneugens were added over a range of concentrations in a volume of 2 μL. A square root 2 dilution scheme was used, and four solvent control wells were included on each 96‐well plate. For these follow‐up experiments, the highest concentration was the same top analyzable concentration from the initial MultiFlow DNA Damage Assay, but in this case, cells were only treated for 4 h, and each concentration was tested in duplicate. The following concurrent controls were studied: carbendazim, AMG‐900, and epothilone A.The test chemicals identified as exhibiting an aneugenic MoA were tested in a follow‐up assay designed to investigate tubulin binding vs. mitotic kinase inhibition activities. First, 488 Taxol and unlabeled Taxol supplied in prototype MultiFlow Aneugen Molecular Mechanism Kits  were added to TK6 cells for final concentrations of 250 and 100 nM, respectively. The cells were then seeded into wells of a U‐bottom 96‐well plate  containing 50 μL/well of pre‐aliquoted lysis/labeling solution from a prototype MultiFlow—Aneugen Molecular Mechanism Kit. The proprietary lysis/labeling solution was used to simultaneously digest cytoplasmic membranes, stain chromatin with a fluorescent nucleic acid dye, and label several epitopes with fluorescent antibodies. Specifically, anti‐Ki‐67‐eFluor 660 was used as a mitotic cell marker, and anti‐phospho‐histone H3‐PE served as a mitotic marker that is responsive to Aurora kinase B inhibition. Kit‐supplied RNase and propidium iodide were used to label chromatin, and a known concentration of latex microspheres  provided a means to calculate nuclei density. After incubation at ambient temperature for 60 min in the dark, samples were analyzed via flow cytometry.n and greater DNA content) and their high‐fluorescing PE and eFluor 660 signals. The 488 Taxol biomarker was based on median channel fluorescence. For all graphical representation and statistical analyses, these values were first converted to mean fold‐change relative to a plate‐specific solvent control arithmetic mean. Gating logic required these events to exhibit propidium iodide–associated fluorescence corresponding to 2n–4n DNA content. Ki‐67‐positive cells were excluded from the median channel fluorescence calculation.Flow cytometric analysis was performed using a MACSQuant® Analyzer 10 flow cytometer with integrated 96‐well MiniSampler device . As described previously . Note that one (1) was subtracted from every biomarker's fold‐change value before AUC calculations were made in order to set the no effect (baseline) value to zero. With this offset in place, AUC values were zero or nearly so in the case of no response, positive in the case of an increase, and negative in the case of a reduction.Unsupervised clustering of the responses from the AMM assay was performed with JMP software (v12.0.1) based on AUC data. To convert the biomarker responses to AUC, feature scaling was applied to every test article concentration to bring the values into the range 0 to 1 . The classification model was designed to categorize chemicals into one of four most likely AMM groups: tubulin destabilizers, tubulin stabilizers, Aurora kinase B inhibitors , or a catch‐all “other” (indeterminate) group. As described by Bernacki et al. , the modTK6 cells continuously exposed to a range of NTP BCE and BC XRM concentrations were observed to exhibit significant induction of MN at the 24 h time point Fig. a,b. BaseTK6 cells were exposed to NTP BCE and BC XRM and evaluated for several biomarkers that are useful for distinguishing between clastogenic and aneugenic MoA. As shown by Figure NTP BCE and BC XRM data were evaluated by an ensemble of three ML prediction algorithms based on a training set of 86 chemicals  inhibition. On the other hand, they did lead to concentration‐dependent reductions to 488 Taxol‐associated fluorescence. Unsupervised clustering, provided as a two‐dimensional dendrogram in Figure Finally, AUC values from the AMM assay were applied to an artificial neural net ML classification model (Bernacki et al. In the United States, botanical dietary supplements are used by ~18% of adults and ~5% of children, according to the 2012 National Health Interview Survey (Black et al. To gain a better understanding of the aneugenic activity of NTP BCE and BC XRM, we tested the same samples that were tested in the MicroFlow and MultiFlow DNA Damage assays in the novel MultiFlow AMM assay. Certain molecular events associated with chromosomal malsegregation have been very well characterized, such as disruption of tubulin polymerization and depolymerization, and inhibition of aurora kinases that are crucial for the orderly progression of mitosis (Lynch et al. in vivo and one that is a vouchered standard reference material, are caused by destabilization of tubulin. In addition, we speculate that destabilization of tubulin could also potentially account for the megaloblastic anemia condition observed in female mice and rats exposed to BCE (Mercado‐Feliciano et al. Taken together, our data strongly suggest that MN induced by two key BCE samples, one that exhibited genotoxic activity The confirmed aneugenic activity of BCE has implications for evaluating the risk of genotoxic damage from exposure to BCE because aneugenic agents are well understood to exhibit nonlinear dose–response relationships (Elhajouji et al. Although BCE exhibits genotoxic potential, the use of BCE should be considered in light of risk–benefit assessments. BCE is marketed to women to alleviate symptoms of gynecological ailments, suggesting that it may contain estrogenic constituents Gafner . HoweverAll authors contributed to experimental design. D.T.B. and S.M.B. performed benchtop work, and S.D.D. performed statistical analyses. S.L.S. and S.D.D. generated a first draft of the manuscript, and all authors contributed to the revisions that followed.In Vitro MicroFlow® Kit and the MultiFlow® DNA Damage Kit—p53, γH2AX, Phospho‐Histone H3, and intends to sell the MultiFlow® Aneugen Molecular Mechanism Kit described herein.D.T.B, S.M.B, J.C.B, and S.D.D are employed by Litron Laboratories. Litron holds patents for flow cytometry‐based analyses described herein. Litron currently sells the


Pomatoschistus minutus, a marine fish with external fertilization, we used a paired experimental design, with spermatozoa tested with and without sperm-duct gland content mixed into seawater. We measured sperm velocity, percentage of motile sperm and sperm viability over time. Sperm were found to swim 7.3% faster when gland content was mixed in the seawater. Percentage motile sperm was unaffected by the gland content. Sperm viability in seawater exceeded 24 h, but was unaffected by the gland content. An increase in sperm velocity of similar magnitude as found here has been shown by others to increase fertilization success. Since velocity-boosting properties of sperm-duct gland content have now been found in three distantly related goby species, this trait appears to be conserved across the Gobiidae family and may aid in reproduction across a range of species and environments.Sperm performance is often tightly linked to male reproductive success. In many demersal gobiid fishes, the male attaches sperm embedded in a mucus produced by sperm-duct glands to the nest substrate before spawning takes place. Sperm are activated as the mucus and embedded gland content dissolve into the water. To test the importance of gland content on sperm function in This article has an associated First Person interview with the first author of the paper. Summary: In sperm, velocity is often traded off for viability. However, we show here that in sand gobies, sperm-duct gland content increase velocity without reducing viability, thus mitigating such a trade-off. Drosophila to humans  between their velocity and viability . This tro humans . DespiteAmong fish, blennies , sculpinPomatoschistus minutus, Pallas 1770, painted goby, P. microps, Krøer 1838, and two-spotted goby, Gobiusculus flavescens, Fabricius 1779, C. Kvarnemo et al., unpublished data).Sperm competition is common among gobies . As femaThe physiological properties of the SDG content was studied during the early days of comparative physiology , but resSupplementary information.For detailed procedures and definitions, please see the Materials and Methods and 2 (1)=0.1374, P=0.7109], detection threshold , or number of tracked sperm , which supports the sampling methodology. To control for a potential effect of sperm numbers on velocity, a general linear model analysis of covariance was performed with VCL as dependent variable, treatment as factor and percent motile sperm as covariate. Non-significant interactions (P>0.05) were deleted from the model. Despite heterogeneous variances, the viability data were analysed with a repeated measures general linear model with both time and treatment as repeated measures for each individual replicate.No difference was found in the percent of motile sperm  (2(1)=0.1374, P=0.7109].We found that the treatment with content from the sperm-duct glands significantly increased the velocity of sperm in the sand goby [tested with SDG content mean±s.e.m. VCL: 75.38±2.07 μm sP<0.001] B. The pe1,9F=8.92, P=0.015; treatment (repeated): 1,9F=0.10, P=0.76; time*treatment: 1,9F=0.03, P=0.87] :  P=0.87] C. In botSperm velocity is directly linked to increased fertilization success in many taxa , and an Zosterisessor ophiocephalus, Pallas 1814) and their close relative, the black goby   .The otherwise short lifespan of fish sperm in general has been attributed to an inability to handle osmotic change when ejaculated into an environment of different osmolality . HoweverAlternative reproductive tactics were not a focus of this study; yet, their role for SDG evolution makes them relevant to discuss in this context. As expected from theory , sneakerZosterisessor and Gobius, which are closely related, and Pomatoschistus belong to two distinct lineages . The SDG adaptations in Gobiidae possibly have an even older origin, as the sister families Butidae, Eleotridae and Odontobutidae also have SDG structures , the ability to influence sperm function in the fertilization micro-environment is of interest for future research, in particular as this factor may contribute to their ability to spread into a range of environments.Our results are thus mirrored in grass and black goby, where SDG content also has a positive effect on sperm performance . Since tlineages , our resructures . Gobiidaructures . Their sructures , togetheructures  points tructures ; Fig. S1Gasterosteus aculeatus, Linnaeus 1758), which has ovarian fluid that enables its sperm to function in a range of salinities  males in were caught in Bökevik, Fiskebäckskil, Sweden (58°14'54.1″N 11°26'48.0″E), ∼10–11 May 2015. Males develop breeding colouration at the start of the breeding season (late April–early May), and in this study, we only used males in breeding colouration, as a way of avoiding immature males, as well as sneaker males, which lack breeding colouration . The sample was then stirred using a Vortex  three times for 1 s in rapid succession. The sperm were then activated by transferring 25 μl of each suspension to a new microcentrifuge tube, filled with 750 μl filtered seawater  using a micropipette  in an effort to mimic the natural way the ejaculate would mix with in the external environment. Thus, we had one tube for each treatment and male that was kept at 10°C in a thermostatic bath during the entire experiment except during vortexing. Testing of the samples was performed identically for both treatments.While a natural ejaculate may contain sperm and seminal fluid produced by the testes, potentially mixed with products of the SDG, we were interested in the specific impact of SDG content on sperm performance. For this reason, and because it is not possible to strip sand gobies without injuring the fish, we used dissections to be able to test this in a controlled way. Sample sizes were kept as low as possible and decided based on previous studies of sperm motility in the species . Sperm f−s (see Table S3 for settings). We measured velocity of the curvilinear path (VCL), velocity of the average path (VAP), velocity of the straight line path (VSL), progression (PROG) and beat cross frequency (BCF). We focused our analysis on VCL, due to its common use in studies on goby sperm velocity (Table S1. (2) Motility was measured as percent sperm that exceeded minimum speed (same as above). (3) Viability was calculated as percentage live sperm (number of live/number of live and dead ×100), measured after 10 min and 24 h.We measured three traits that relate to sperm performance: velocity, motility and viability. Here we define them as follows: (1) velocity (or swimming speed) was only measured for sperm swimming at a minimum speed of 25 μmvelocity , includivelocity  and becavelocity  includinvelocity . Results−1, size 2592*2048 pixels, exposure time 10 ms, Gain 0, Gamma 0.1). All handling was done as quickly as possible and always <10 min after dissection.To assess sperm velocity and percentage of motile sperm, the sample for each male and treatment was stirred using a Vortex-Genie 2 (same model as above) 3×1 s to mix it. From this, three 45 μl suspensions were transferred to an albumin coated microscope slide fitted with three O-rings. Each drop was covered with an albumin coated coverslip to form a suspended drop following established protocols . This pr® Sperm Viability Kit, L7011, Life Technologies, Thermo Fisher Scientific) (To investigate sperm viability the following procedure was done 10 min (±1 min) after organ dissection and 24±1 h later for each treatment: 100 μl was taken from the sample for each male and treatment and transferred to a separate 600 μl microcentrifuge tube  and vortexed for 3×1 s. This sample was stained with 1 μl diluted SYBR14 (1 parts SYBR14 to 49 parts DMSO) and then stained with propidium iodide (diluted one parts propidium iodide to four parts with DMSO) (LIVE/DEADentific) . The samTable S3 for details on settings used. An average of 282 sperm cells per film were tracked.Images were digitally filtered and analysed using ImageJ  and videos were analysed using a computer assisted sperm analysis plugin (CASA) , 2011. SF1,19=7.23, P=0.015) or VCL . Sperm motility data were analysed by linear mixed effects models, using ‘treatment’ (sperm with or without SDG content) as fixed factor and controlling for individual variation by including ‘individual’ and ‘technical replicate’ as random factors. To obtain the effect size estimate , a likelihood ratio test was then used to compare two mixed effects models, one with and the other without the ‘treatment’ factor.Data were statistically analysed using SPSS 22 (IBM) and R . All data were tested for outliers, normal distribution and homogeneity of variances and covariance. There were no outliers in the data (as assessed by box-plot) and data was normally distributed  but assumptions of homogeneity of variance were not met for viability (Levene's Test,


We evaluated the impact of MTH methods on neurological outcomes. Ninety-seven patients who received MTH were included in the present analyses. The neurological outcomes were compared among subgroups of patients divided by cutoff values for the induction, maintenance, and rewarming times of MTH in all patients, in patients with diffuse injury, and in patients with an evacuated hematoma. The proportion of patients with a good neurological outcome was significantly different between patients with an evacuated hematoma divided into subgroups by the cutoff value of rewarming time of 48 h . Slow rewarming for >48 h might improve the neurological outcomes of prolonged MTH in patients with TBI and an evacuated hematoma. Further studies are needed to investigate the optimal rewarming protocol in patients with TBI.Mild therapeutic hypothermia (MTH) is expected to improve the neurological outcomes of patients with severe traumatic brain injury (TBI). However, there are no standard protocols for managing the temperature of patients with severe TBI in order to improve their neurological outcomes. We conducted a  However, there are no standard protocols for managing the temperature of patients with severe TBI in order to improve their neurological outcomes. Nevertheless, several post hoc analyses of major RCTs suggested that pre-surgical induction of MTH potentially improved the neurological outcomes in patients who had evacuation of a hematoma4.Although mild therapeutic hypothermia (MTH) has long been expected to improve the neurological outcomes of patients with severe traumatic brain injury (TBI), two major randomized controlled trials (RCT) did not find any advantages of MTH in this setting5, but MTH (32.5 °C) for 24 h did not have positive results in children with TBI6.To complicate the situation, different MTH protocols are used in patients with other forms of brain damage, and these have yielded different outcomes. For example, MTH (33.5 °C) for 72 h was associated with favorable neurological outcomes in neonates with hypoxic encephalopathy7. Although the B-HYPO study found no beneficial effect of MTH (32.0–34.0 °C) compared with fever control (34.5–37.0 °C) in neurological outcome, MTH had statistically significant benefits in neurological outcome of patients with an evacuated hematoma4. Therefore, we re-analyzed the data from the MTH group to evaluate the impact of the durations of the induction, maintenance, and rewarming phases on the neurological outcomes of patients with TBI.The Brain Hypothermia (B-HYPO) study was a Japanese RCT of MTH for severe TBI, in which the temperature management protocol involved a rapid induction phase (≤6 h), a prolonged maintenance phase (≥3 days), and a slow rewarming phase (<1.0 °C/day)post hoc analyses of patients in the MTH group enrolled in the B-HYPO study. The B-HYPO study was performed between December 2002 and September 2008. It was designed as a multicenter RCT with prospective analyses and blinded assessment of neurological outcomes. The protocol and consent procedures were approved by the Institutional Review Board of each participating hospital , and all experiments were performed in accordance with relevant guidelines and regulations. The study was registered with the University Hospital Medical Information Network (UMIN C000000231 (13/09/2005): https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000000293) in Japan and the National Institutes of Health : https://clinicaltrials.gov/ct2/show/NCT00134472?cond=NCT00134472&cntry1=ES%3AJP&rank=1) in the United States of America. The written informed consents were obtained from all inclusion cases. The randomization list was automatically generated by the UMIN computer system to allocate patients in a 2:1 ratio to receive MTH (32–34 °C) or fever control (35.5–37 °C). Males and females were eligible if they were aged 15–69 years, had a Glasgow Coma Scale (GCS)8 score of 4–8, and cooling could be started <2 h after the onset of TBI. Patients with any of the following were excluded: good motor response (GCS motor response score 6), systolic blood pressure <90 mmHg after fluid and vasopressor resuscitation, platelet count <50,000 /mm3, severe pre-existing medical conditions , acute myocardial infarction, pregnancy, severe alcohol intoxication that prevented assessment of consciousness, penetrating brain injury, epidural hematoma without brain parenchymal injury, or core body temperature <30 °C. Eligible patients were enrolled and their core temperature was lowered to the target temperature as quickly as possible. Computed tomography (CT) of the head was performed and evaluated on admission and after rewarming in all patients.We conducted 2, systemic vascular resistance index (SVRI) 800–1200 dynes/s/cm5, ICP < 20 mmHg, and cerebral perfusion pressure >60 mmHg. The partial pressures of arterial oxygen and carbon dioxide were maintained at >100 mmHg and 30–40 mmHg, respectively. If ICP was >20 mmHg, any treatment recommended by the Japanese guidelines9 could be administered, including mannitol/glycerol and/or a bolus infusion of barbiturates. Continuous infusion of barbiturates was prohibited due to its severe suppression of cardiac function. Hyperventilation was allowed, but excessive hypocapnia <30 mmHg was prohibited. The ICP could also be reduced by surgical removal of the patient’s cranial bone flap. Anti-convulsants were allowed as deemed necessary by the attending physician.The core body temperature was measured by a thermister coupled to an internal jugular venous catheter. If the catheter could not be inserted at this site, body temperature was measured at another site that was selected in the following order: pulmonary artery, bladder, rectum, and tympanic membrane. In addition to critical neurological care, an arterial catheter, a pulmonary arterial catheter, and an intracranial pressure (ICP) monitoring probe were inserted to monitor and maintain the patient’s hemodynamic status and ICP at the following levels: mean arterial pressure >80 mmHg, cardiac index >2.5 L/min/mCooling blankets, rapid cold fluid infusion , and/or cold gastric lavage could be used during the induction phase in both groups. The aim was to achieve the target temperature <6 h after the onset of TBI. The target temperature was to be maintained for ≥72 h, mainly using surface cooling blankets, in both groups. The patient was rewarmed at a rate of <1 °C/day and the core body temperature was maintained at <38 °C for 7 days after the onset of TBI.Sedatives and analgesics were usually tapered off once the patient had been rewarmed to 36 °C. The muscle relaxant was stopped when the patient had stopped shivering, which usually occurred during the maintenance phase. Muscle relaxants were restarted as deemed necessary by the attending physician.10. Hemodynamic and laboratory data were recorded on Days 0, 1, and 3, as well as 1 day after rewarming (defined as the day on which the core body temperature reached 36 °C). Clinical values were recorded for each day.All data, except the head CT data, were sent to the UMIN center via an internet-based system. The injury severity score (ISS) and head CT classification were also assessed radiologists and neurosurgeons. In this study, the ISS was also calculated as an abbreviated injury score (AIS). The head CT was classified as follows: diffuse injury grade I, all diffuse injuries without CT findings; diffuse injury grade II, high or mixed density lesions with a volume of <25 ml; diffuse injury grade III, high or mixed density lesions with a volume of <25 ml and compressed or absent basal cisterns; diffuse injury grade IV, high or mixed density lesions with a volume of <25 ml and a midline shift of >5 mm; and evacuated/non-evacuated hematoma, high or mixed density lesions with a volume of >25 ml with or without surgical evacuation11 at 6 months, and was assessed by a neurosurgeon, neurologist or emergency physician unaware of the patient’s treatment method. One month and 6 months after discharge from the original hospital, the patients were followed-up by telephone. The neurological outcomes were classified as good (moderate disability or good recovery) or poor .The primary outcome was the Glasgow Outcome Scale (GOS)In the present study, we compared the proportion of patients with good neurological outcomes between subgroups of patients divided by cutoff values for the induction phase (time taken to reach 34.0 °C ≤6 h), maintenance phase (duration of hypothermia ≥72 h), and rewarming phase (time taken to rewarm to 36.0 °C ≥48 h) in all patients, in patients with diffuse injury, and in patients with an evacuated hematoma. These cutoff values of induction (≤6 h), maintenance (≥72 h), and rewarm (≥48 h), were considered as ideal target of therapeutic hypothermia for severe TBI by our study group and protocol.U test. Categorical variables were statistically analyzed using Fisher’s exact probability test. Multivariate analysis was performed by logistic regression analysis. The threshold of significance was set at P < 0.05. Statistical analyses were performed using SPSS software version 23.0 .Continuous variables were statistically analyzed using the Mann–Whitney Showa University Hospital, Tokyo; Yamaguchi University Hospital, Yamaguchi; Japanese Red Cross Musashino Hospital, Tokyo; Sapporo University Hospital, Hokkaido; Kagawa University Hospital, Kagawa; Osaka Mishima Emergency Critical Care Center, Osaka; Gifu University Hospital, Gifu; Nippon Medical School Hospital, Tokyo; Ehime University Hospital, Ehime; Fujita Health University Hospital, Aichi; Chiba Emergency Medical Center, Chiba; St. Marianna University Hospital, Kanagawa; Saiseikai Utsunomiya Hospital, Tochigi; Kitasato University Hospital, Kanagawa; Kawasaki Medical School Hospital, Okayama; Nara Medical University Hospital, Nara; Chiba University Hospital, Chiba; Nagoya Medical Center, Aichi; The Hospital of Hyogo College of Medicine, Hyogo; National Tokyo Medical Center, Tokyo; Nihon University Hospital, Tokyo; Nippon Medical School Tama Nagayama Hospital, Tokyo; Tokyo Medical University Hachioji Medical Center, Tokyo; Tokyo Medical University Hospital, Tokyo; Aizawa Hospital, Nagano; Shinshu University Hospital, Nagano; Shiga University Hospital, Shiga; Tokushima University Hospital, Tokushima; Toho University Omori Medical Center, Tokyo; Teikyo University Hospital, Tokyo; Saitama Medical Center, Saitama; Osaka National Hospital, Osaka; Kansai Medical University Takii Hospital, Osaka; Ohota Nishinouchi Hospital, Fukushima; Oita University Hospital, Oita; and Iwate Medical University Hospital, Iwate. All collaborating hospitals are in Japan.Table P = 0.0498). This suggests that slow rewarming  may confer better neurological outcomes than quicker rewarming.Tables P < 0.0498) and the mortality rate (P = 0.015) were significantly different between these two groups of patients. There were also some differences, albeit not significant, between the two groups, with age, GCS score on admission, duration of the hypothermia maintenance phase, and initial ICP being younger, more severe, longer, and lower, respectively, in the ≥48 h rewarming group than in the <48 h rewarming group.Table Table post hoc analyses were performed under our desire to improve the temperature management of MTH, because the B-HYPO study involved a long duration of hypothermia  and a long rewarming time  owing to the slow rewarming rate of ≤1.0 °C/day in a well-controlled study in terms of age, GCS, hemodynamic control, and inter-/intra-center randomization. We previously reported that patients with an evacuated hematoma experienced better neurological outcomes after MTH than patients with diffuse brain injury4. Therefore, we re-analyzed the results of the MTH group in patients with an evacuated hematoma or diffuse injury in the B-HYPO study.The present 13, but there was no clinical evidence, except for a systematic review and a guideline to clearly support this approach in humans15. Usually, the clinical protocols for MTH have focused on the rate of cooling and the duration of MTH, rather than the duration or rate of rewarming. Therefore, we re-analyzed the MTH group of the B-HYPO study to examine the relative impact of each phase  on neurological outcomes using clinically appropriate cutoff values for each phase. We found that a rewarming phase of ≥48 h  was associated with a significantly greater rate of good neurological outcomes and significantly lower mortality rate compared with a rewarming period of <48 h  in patients with an evacuated hematoma  was associated with better neurological outcomes, and the authors hypothesized that hypothermia suppressed rebound intracranial hypertension during prolonged hypothermia and rewarming16. Although a reduction of ICP by MTH did not improve the neurological outcomes of patients with TBI17, animal studies revealed that MTH suppressed TBI-related brain damage in both diffuse injury and post hematoma evacuation18. Nevertheless, recent clinical studies reported that the advantage of MTH on neurological outcomes was only apparent in patients with severe brain injury and an evacuated hematoma4. These results imply that the neurological outcomes after MTH are dependent on the type of brain damage.In the present study, we found no significant effects of MTH in terms of the induction time (≤6 h) and duration of hypothermia (≥72 h). The induction times analyzed here are similar to those used in two earlier RCTs (8.4 h and 4.4 h)20. The mean (range) initial ICP were 14 mmHg (7–40 mmHg) and 67 mmHg (20–92 mmHg) with rewarming times of ≥48 h and <48 h, respectively , in 3 patients during the maintenance phase, in 3 patients before rewarming, and in 2 patients after rewarming, in the sub-group of patients with a rewarming time of <48 h. These results are similar to those of earlier studies20. We hypothesize that ischemia/reperfusion of the injured brain tissue might cause recurrent ICP elevation/reduction during the rewarming phase. In the present study, blood flow to the injured brain regions was adequate based on the cardiac index  might be associated with better neurological outcomes than quicker rewarming  in patients with TBI and an evacuated hematoma. Our results also suggest that prolonged rewarming of ≥ 48 h together with frequent measurement of ICP might be an appropriate strategy for preventing rebound intracranial hypertension in patients with TBI and an evacuated hematoma.


AAA) is unknown. We aimed to evaluate the relationship between adherence to a Dietary Approaches To Stop Hypertension‐style dietary pattern and the risk of incident AAAs.The role of a healthy dietary pattern in the prevention of abdominal aortic aneurysms . A dietary scoring index based on food times was constructed to assess self‐reported adherence to a dietary approaches to stop hypertension‐style dietary pattern. Participants were followed for incident clinical AAAs using hospital discharge diagnoses, Medicare inpatient and outpatient diagnoses, or death certificates through December 31, 2011. Cox proportional hazards models with covariate adjustment were used to estimate hazard ratios with 95% confidence intervals. During a median follow‐up of 23 years, there were 517 incident AAA cases. Individuals with a Dietary Approaches To Stop Hypertension‐style diet score in the highest quintile had a 40% lower risk of hospitalization for AAA than those in the lowest quintile . In detailed analyses, higher consumption of fruits, vegetables, whole grains, low‐fat dairy, and nuts and legumes was related to a lower risk for AAA.Dietary intake was assessed via a 66‐item food frequency questionnaire at baseline (1987–1989) and at visit 3 (1993–1995) in 13 496 participants enrolled in the ARIC (Atherosclerosis Risk in Communities) study without clinical AAA. Higher consumption of fruits, vegetables, whole grains, low‐fat dairy as well as nuts and legumes may help to decrease the burden of AAAs.Greater adherence to a Dietary Approaches To Stop Hypertension‐style dietary pattern was associated with lower risk for  The role of a healthy dietary pattern in the prevention of abdominal aortic aneurysms is largely unknown.In a community‐based setting, individuals with high adherence to a Dietary Approaches To Stop Hypertension‐style dietary pattern had a 40% lower risk of hospitalization for abdominal aortic aneurysm than those with low adherence.Higher consumption of fruits, vegetables, whole grains, low‐fat dairy as well as nuts and legumes may help to decrease the burden of abdominal aortic aneurysms in US adults.Abdominal aortic aneurysms (AAAs) develop in up to 8% of men and 6% of women over a lifetime.The Dietary Approaches to Stop Hypertension (DASH) diet is a healthy and balanced dietary pattern currently promoted by the National Heart, Lung, and Blood Institute of the National Institutes of Health, the United States Department of Agriculture, and the American Heart Association/American College of Cardiology as a healthy diet for all Americans.The objective of this study was to assess if adherence to a DASH‐style dietary pattern is associated with an altered risk for AAA. We therefore examined the association between adherence to DASH‐style dietary pattern and its components with subsequent incidence of AAA in a large, US community‐based cohort of middle‐aged adults. We hypothesized that adherence to a DASH‐style dietary pattern would be associated with a lower risk for AAAs. Second, given previous data suggesting a close linkage between chronic inflammation and the development of AAA,The data, analytic methods, and study materials are made available to other researchers for purposes of reproducing the results or replicating the procedure. The data underlying our work can be obtained through 2 mechanisms. First, interested investigators can contact the ARIC Coordinating Center at the University of North Carolina—Chapel Hill. Details about the procedures for data request can be found online.The ARIC (Atherosclerosis Risk in Communities) study is a community‐based prospective cohort study of 15 792 middle‐aged adults (aged 45–64 years at baseline) from 4 US communities .All participating institutions received institutional review board approval for the study and all participants provided written informed consent.The ARIC study assessed dietary intake using a 66‐item food frequency questionnaire (FFQ) adapted from the 61‐item FFQ developed by Willett et alTwo previously developed dietary scoring indices based on food items or nutrient targets were applied to assess adherence to a DASH‐style diet.In sensitivity analyses, we applied the DASH diet score based on nutrient targets. This score consisted of 9 components, ie, low intake of (1) saturated fat, (2) total fat, (3) cholesterol, and (4) sodium and high intake of (5) protein, (6) fiber, (7) magnesium, (8) calcium, and (9) potassium.Both scoring systems were analyzed using quintiles with a higher quintile reflecting higher adherence to a DASH‐style diet. We also analyzed quintiles of individual food and nutrient components (including sodium/potassium ratio) of the DASH diet scores. Study participants did not receive dietary counseling, and the DASH‐style diet results were not published at the time of ARIC study visits 1 and 3.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD‐9‐CM) codes of 441.3 or 441.4, or procedure codes of 38.44 or 39.71, or the following cause of death codes: ICD‐9 441.3 or 441.4 or ICD‐10 code I71.3 or I71.4.The primary end point for this study was incident AAA after baseline . Incident clinical AAAs were identified by searching hospitalization and death records as well as Medicare data as previously outlined.Covariates were assessed at baseline via standardized protocols or questionnaires.2), abdominal obesity , hypertension (yes/no), diabetes mellitus (yes/no), hypercholesterolemia (yes/no), and cardiovascular disease (yes/no) .Means and proportions were used to describe baseline characteristics  according to quintiles of the food‐based DASH‐style diet score. To assess the crude (unadjusted) association of the DASH‐style diet score with AAAs, we calculated incidence rates (IR) of incident AAA per 1000 person‐years as the number of diagnosed cases of AAA occurring during the entire follow‐up period divided by person‐years of follow‐up according to quintile of the DASH‐style diet score. Time to event was defined as time from the baseline examination to the date of AAA identification, death, lost to follow‐up, or December 31, 2011. Cox proportional hazards regression models were used to account for potential confounding on the association between the DASH‐style diet score and incident AAAs. An initial model adjusted for sex, total energy intake, race‐center, and age by the use of restricted cubic splines with knots at 45, 51, 57, and 63 years of age representing the 5th, 35th, 65th, and 95th percentiles . A second model adjusted for all of the baseline covariates in model 1 plus alcohol intake quintiles (sex specific quintiles of g/week), education , household income , smoking status , sport‐related physical activity index, leisure‐time physical activity index, body mass index category  after fully adjusting for confounding variables (model 2). These main results did not change substantially when we excluded current smokers at baseline from our analyses  (Table Pinteraction by sex=0.32), race (Pinteraction by race=0.38), smoking (Pinteraction by smoking=0.79), or obesity (Pinteraction by obesity=0.16).A total of 517 cases of incident hospitalized AAA cases were observed during a median follow‐up of 23 years among 13 496 participants. Across quintiles of increasing DASH dietary scores based on food items, individuals with greatest self‐reported adherence had a lower risk for incident AAA hospitalizations (Table es Table  or underes Table . Further6) Table . When weQ5 vs Q1: 0.70; 95% CI: 0.52, 0.95, Ptrend=0.013; vegetables HRQ5 vs Q1: 0.60; 95% CI: 0.44, 0.80, Ptrend<0.001; whole grains HRQ5 vs Q1: 0.67; 95% CI: 0.51, 0.89, Ptrend=0.027; low‐fat dairy HRQ5 vs Q1: 0.48; 95% CI: 0.36, 0.63, Ptrend<0.001). In addition, higher nuts and legumes consumption was associated with reduced AAA risk . Higher sodium intake was significantly associated with a decreased risk for incident AAAs , whereas a higher sodium‐potassium ratio was not associated with AAA risk . Dietary intake of red and processed meat products as well as sweetened beverages were not significantly associated with AAA risk in the fully adjusted model. In detailed analyses of nutrient‐item components of a DASH‐style dietary pattern, lower saturated fat, higher protein, and higher fiber intake were associated with a lower risk for incident AAAs .To assess for partial mediation of the association between the DASH style dietary pattern and incident AAA risk, we additionally adjusted for hypertension modeled as a time‐varying variable. Our results changed only marginally Table . After fIn a large, community‐based cohort of middle‐aged adults, greater self‐reported adherence to a DASH‐style dietary pattern was associated with a significantly lower risk of AAA hospitalization.The majority of the prospective epidemiological evidence for a dietary association with incident AAAs stems from studies of dietary components involving individuals from Sweden and populations in the United States.Our results for a heart healthy dietary pattern known to lower blood pressure extend previous reports on single food items suggesting that adherence to a healthy, DASH‐style eating plan can significantly decrease AAA risk. In our community‐based study population, a diet rich in vegetables, whole grains, low‐fat dairy products, and nuts and legumes shows promising results for reducing the risk of incident AAAs. On a nutrient basis, higher saturated fat intake was related to greater risk for AAA which is in line with previous experimental data,Hypertension was not a strong mediator of the observed inverse association between adherence to a DASH‐style dietary pattern and incident AAA. This underscores, on the one hand, the controversial role of hypertension in the expansion of the AAAs, while it, on the other hand, suggests the presence of other explanatory factors.These results have both clinical and scientific implications. Rather than individual food items, a DASH‐style eating plan, which includes a variety of healthy food choices, may protect against incident AAAs. Second, this study strengthens the concept of the beneficial immuno‐modulating effects of a healthy dietary pattern on inflammatory processes.ICD codes. Although the AAA codes seem specific, we tried to enhance the validity of our AAA outcome by excluding aortic aneurysms coded to uncertain sites because they may not have been abdominal.The strengths of our study include a large diverse, community‐based, prospective cohort with long follow‐up, structured assessment of dietary intake, assessment of and adjustment for relevant covariates, and adjudicated outcome events. Nonetheless, there are several limitations. Our exposure, food intake, was assessed in the late 1980's and mid 1990's; thus, changing dietary habits and food supply over time may not have been adequately captured by self‐reported FFQs. Furthermore, although the nutritional data derived from these FFQs have been shown to be reliable, assessing true dietary adherence to a DASH dietary pattern over time was not possible in our study setting.In conclusion, greater self‐reported adherence to a DASH dietary pattern was found to be associated with lower risk of incident AAA.The Atherosclerosis Risk in Communities study has been funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, under contract numbers HHSN268201700001I, HHSN2682017000021, HHSN268201700003I, HHSN268201700004I, and HHSN268201700005I. Dr Selvin was supported by grants from the National Institutes of Health and National Institute of Diabetes and Digestive and Kidney Diseases (K24DK106414 and R01DK089174). Dr Tang was supported by a grant from the National Heart, Lung, and Blood Institute (R01 HL103695). Dr. Rebholz is supported by a mentored research scientist development award from the National Institute of Diabetes and Digestive and Kidney Diseases (K01 DK107782). Open access publishing was funded by the German Research Foundation (DFG) and the University of Würzburg.None.Table S1. Association of Quintiles of Food‐Based DASH Diet Score With Incident AAA Excluding Current Smokers*Table S2. Association of Quintiles of Food‐Based DASH Diet Score With Incident AAA by Sex*Table S3. Association of Quintiles of Nutrient Based DASH Diet Score With Incident AAA*Table S4. Association of Individual Nutrients of the Nutrient‐Based DASH Diet Score With Risk for AAA*Click here for additional data file.


As the debate continues about whether obesity in metabolically healthy individuals is associated with poor outcomes or not, investigating the association between the obesity phenotypes and markers of subclinical myocardial injury will help identify those at risk for future cardiovascular events (cardiovascular disease [CVD]).We hypothesize that obesity phenotypes including metabolically healthy obesity (MHO) is associated with subclinical myocardial injury (SC‐MI).2.This analysis included 3423 participants  without known CVD from National Health and Nutrition Examination Survey (NHANES) III. Multivariable logistic regression models were used to examine the cross‐sectional association between four obesity phenotypes  [reference], metabolically unhealthy nonobese (MUNO), MHO, and metabolically unhealthy obese (MUO) with SC‐MI. SC‐MI was defined from the 12‐lead electrocardiogram as cardiac infarction/injury score ≥ 10 units. Metabolic syndrome (MetS) was defined according to the International Diabetes Federation consensus definition. Obesity was defined as body mass index ≥30 kg/mP = 0.0005). This association was stronger in men vs women (OR [95% CI]: 2.20 [1.58‐2.07] vs 1.08 [0.79‐1.48]), respectively; interaction P‐value = 0.002) but was consistent in subgroups stratified by age and race. There was no significant association of MHO or MUNO with SC‐MI compared with MHNO, but there was a trend toward higher odds of SC‐MI in the MUNO group .MUO was associated with higher odds of SC‐MI compared with MHNO (odds ratio [OR], 1.53; 95% confidence interval [CI], 1.22‐1.92, Our findings suggest that a combination of obesity and MetS confers worse prognosis and early preventive strategies aimed at weight loss and management of MetS components may decrease the risk of future poor outcomes. One such marker is the cardiac infarction/injury score (CIIS), an electrocardiographic‐based scoring system used to define subclinical myocardial injury (SC‐MI).22.1NHANES is a periodic survey of a representative sample of the civilian noninstitutionalized US population. Its principal aim is to determine estimates of disease prevalence and health status of the US population. The National Center for Health Statistics of the Center for Disease Control and Prevention Institutional Review Board approved the protocol for NHANES‐III. All participants gave written informed consent. Baseline data were collected during an in‐home interview and a subsequent visit to a mobile examination center between 1988 and 1994. The following characteristics were self‐reported: age, sex, race/ethnicity, income, prevalent CVD, cancer history, smoking status, and leisure time physical activity. Medication history, including the use of antihypertensive agents and lipid‐lowering therapies, also were self‐reported. A physical examination was performed to obtain body mass index (BMI). Blood pressure readings were taken during the in‐home evaluation and again during the mobile examination center visit and averaged for each participant. Fasting blood samples were collected to measure total cholesterol, high‐density lipoprotein cholesterol, triglycerides, and glucose, using laboratory procedures as reported by NCHS.For this analysis, we only included NHANES‐III participants who underwent an ECG recording (n = 8561). We excluded participants with a history of CVD , any major electrocardiographic abnormalities based on Minnesota Code classification,2. Obese and nonobese participants were subsequently divided into two subgroups based on the presence of MetS. MetS was defined according to the International Diabetes Federation consensus definition as having three or more metabolic abnormalitiesObesity was defined as BMI ≥ 30 kg/m2.2Resting 12‐lead electrocardiograms were obtained with a Marquette MAC 12 system  during the mobile examination visits by trained technicians. Analysis of electrocardiograms was achieved through a computerized automated process and visual inspection by a trained technician located in a centralized core laboratory. The derivation of the CIIS and methodology have been described previously.2.3t‐test was used to compare the continuous variables while χ2 was used to compare the categorical variables. Multivariable logistic regression analysis was used to compute odds ratios (ORs) and 95% confidence interval (CI) for the cross‐sectional association between each obesity phenotype  and SC‐MI. We calculated P for trend across obesity phenotypes using a multivariable logistic regression model. Two incremental models were constructed: model 1 adjusted for age, sex, race (non‐whites), and socioeconomic status. Model 2 was further adjusted for smoking, physical activity and low‐density lipoprotein (LDL‐C).Baseline characteristics were compared across nonobesity (MHNO and MUNO) and obesity (MHO and MUO). Continuous variables were reported as mean ± SD, while categorical variables were reported as frequency and percentage. Student We also conducted a subgroup analysis of the association between obesity phenotypes and SC‐MI stratified by age (dichotomized at 65), sex and race (white vs non‐white). The models were adjusted similarly to model 2 as mentioned above.Additional analyses were performed as follows: First, we performed multivariable linear regression analysis with each obesity phenotype  as the independent variable and CIIS as the continuous outcome variable to calculate the adjusted mean ± SE. Models were adjusted as mentioned above. Secondly, to assess whether increasing BMI is associated with higher CIIS, we examined the association of BMI categories (18.5‐24.9 (reference), 25‐29.9, 30‐34.9, 35‐39.9, and ≥ 40) with CIIS using linear regression models. Model 1 was adjusted for age, sex, non‐whites, socioeconomic status and model 2 adjusted for model 1 plus smoking, physical activity, LDL‐C, and all MetS components.P‐values were considered significant if less than 0.05.All statistical analyses were performed using with SAS version 9.4 , and 3This analysis included 3423 participants  of whom 49.6%, 21.8%, 9.6%, and 18.8% had MHNO, MUNO, MHO, and MUO, respectively. SC‐MI was present in 21.7% (n = 744) of participants. The prevalence of SC‐MI was 19.3% in MHNO, 24.3% in MUNO, 16.9% in MHO and 27.4% in MUO participants. Table Among obesity group, MUO were more likely to be old, men, white, smokers, and to have low annual income and physical activity levels compared to MHO. Among the nonobesity group, MUNO were more likely to be old, women, white, nonsmokers, and to have low annual income and physical activity levels compared to MHNO.P = 0.0005). There was no statistically significant association between MHO or MUNO with SC‐MI; However, a trend of higher odds of SC‐MI was observed in MUNO  . There was no statistically significant interaction by age or race .Table 4We examined the cross‐sectional association between obesity phenotypes and SC‐MI using data from NHANES III. The key findings of our study are as followsThese results taken together indicate that the combination of obesity and MetS is associated with higher odds of SC‐MI and mean values of CIIS. BMI and other measures of adiposity have been consistently associated with CVD, and the major impact of obesity on cardiovascular health is mediated by accompanying metabolic abnormalities.MHO is considered a transient state; the duration and severity of obesity leads to an unhealthy state with the passage of time, thus increasing the risk for CVD events.We observed a strong association of obesity phenotypes with SC‐MI among men. Men generally have a higher incidence of CHD and higher age‐adjusted CHD mortality rate compared to women.In previous studies, MUNO had similar CVD risks as those with MUO.Our study has certain limitations. First, we are unable to establish a temporal relationship between obesity phenotypes and SC‐MI due to the cross‐sectional design of the study; However, it is unlikely that SC‐MI leads to obesity and Mets, but the opposite is more plausible. Some of the measurements such as smoking and physical activity are self‐reported and thus subject to recall bias. Finally, we adjusted for several confounders, but residual confounding remains a possibility. Strengths of the study include a large sample size and a community‐living multiracial population with generalizability to the US population, as well as the fact that the key variables were ascertained using standard protocols.5Our results in NHANES‐III provide evidence that MetS may contribute to myocardial injury especially in those with obesity. Also, the higher the obesity class based on BMI, the higher is the risk of myocardial injury independent of MetS. With the increasing prevalence of obesity and MetS, it is important to identify high‐risk populations so that finite resources can be allocated to appropriate groups.The authors have no conflicts of interest to report.Table S1. Definition of metabolic syndrome and obesity phenotypes.Table S2. Association of cardiac infarction/injury score (CIIS) with body mass index (BMI) categories.Click here for additional data file.


Lycopodium clavatum L.), camellia (Camellia sinensis L.), cattail (Typha angustifolia L.), and dandelion . Dynamic image particle analysis (DIPA) and field-emission scanning electron microscopy (FE-SEM) were used to investigate the morphological characteristics of the capsules, and Fourier-transform infrared (FTIR) spectroscopy was used to evaluate their chemical properties. We found that SDMCs undergo bulk degradation in a species-dependent manner, with camellia SDMCs undergoing the most extensive degradation, and dandelion and lycopodium SDMCs being the most robust.Sporoderms, the outer layers of plant spores and pollen grains, are some of the most robust biomaterials in nature. In order to evaluate the potential of sporoderms in biomedical applications, we studied the biodegradation in simulated gastrointestinal fluid of sporoderm microcapsules (SDMCs) derived from four different plant species: lycopodium ( In a broad sense, the structures and compositions of empty sporoderms, which are sporoderm microcapsules (SDMCs) obtained from different species of plants, are generally similar, composed of an inner (or intine) layer of pectin, cellulose and hemicellulose and an outer (or exine) layer of highly robust sporopollenin6. In addition to exhibiting favorable species-specific properties such as uniform architectures, narrow size distributions and highly consistent chemical compositions7, the natural abundance of pollens makes SDMCs a subject of significant interest, especially as natural materials for the microencapsulation of therapeutic agents such as drug compounds17, proteins23, peptides24, nucleotides25, nanoparticles27, and even living cells28.Sporoderms of pollens are some of the most durable and complex materials in nature owing to their distinct layered structures and chemical properties30, and a growing body of evidence has indicated that intact SDMCs enter the bloodstream and lymph system through the intestinal wall32. For example, Atwe et al. investigated the effects of orally administered ovalbumin-loaded lycopodium SDMCs in mice, reporting the resultant production of large amounts of anti-ovalbumin IgG in the serum30. Their results evidenced the suitability of SDMCs as protein carriers that can effectively shield their payloads from the acidic environment of the gastrointestinal tract. In their study, they also demonstrated the ability of intact SDMCs to cross the intestinal barrier and release their contents into the circulatory system, bringing about systemic effects in the model organism.The potential use of SDMCs as carriers for orally administered drug compounds has been previously investigated using lycopodium sporoderm capsules31, the release of the contents into the blood plasma has been shown to occur via an enzymatic pathway, leading to the degradation of the SDMCs31. For example, lycopodium SDMCs loaded with the drug 3,4-diaminopyridine and administered orally to botulism-intoxicated mice were shown to exhibit drug release via a diffusion pathway that is stimulated by intestinal pH conditions33.While persorption, endocytosis and macrophage engulfment have been proposed as plausible mechanisms for gastrointestinal SDMCs uptakeThese findings have great significance for the potential use of SDMCs as drug carriers. However, the biological fate of SDMCs is yet to be fully understood, mainly because the details of the chemical compositions and degradation mechanisms of SDMCs upon oral uptake remain elusive.e.g., ketones, esters, carboxyls and ethers)37 that constitute sporopollenin can lead to differences in the degradation profiles of the resultant SDMCs. Accordingly, addressing this issue would improve our fundamental understanding of sporopollenin chemistry in biological systems, whose knowledge is indispensable for perfecting SDMCs as drug carriers and extending their application to other biomedical fields.Despite being fundamentally similar, sporopollenin from different plant species exhibit significant structural, compositional and property differences owing to their divergent phylogenetic origins. Furthermore, variations in the complex matrix of lipids, carbohydrates and other oxygenated species (Lycopodium clavatum L.) of the lycopodiophyte family, camellia (Camellia sinensis L.) and dandelion  of the dicots family, and cattail (Typha angustifolia L.) of the monocots family38—using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) as a model for the conditions of the human digestive system. These four species were selected because they are commercially harvested and thus easily obtained. Dynamic image particle analysis (DIPA) and field-emission scanning electron microscopy (FE-SEM) were used to investigate the morphologies of the SDMCs, and Fourier-transform infrared (FTIR) spectroscopy was used to investigate the chemical bonds in the SDMCs. We believe that the findings reported herein are invaluable for understanding the degradation mechanism of different SDMCs in biological systems, as well as constituting a scientific basis for the future biomedical application of SDMCs.In this study, we investigated the degradation of SDMCs derived from four different plant species—lycopodium (Lycopodium clavatum spores (S-type) were purchased from Sigma-Aldrich Co., LLC . Camellia sinensis (camellia) bee pollen was purchased from Xi’an Yuenun Biological Technology Co., Ltd. . Natural Typha angustifolia (cattail) pollen grains were sieved from cattail flowers purchased from Wong Yiu Nam Medical Hall Pte. Ltd. (Singapore). Defatted Taraxacum officinale (dandelion) pollen grains were purchased from Greer Labs . All solvents and reagents were obtained from Sigma-Aldrich Co., LLC, nylon mesh was purchased from ELKO Filtering Co., LLC  and 50 µm Duke Standards polystyrene microspheres were purchased from Thermo Fisher Scientific Pte. Ltd. .Natural 18.The preparation method of SDMCs from camellia, cattail and dandelion was the same as that previously reported for lycopodium with slight modificationsL. clavatum spores (100 g) were defatted by refluxing in acetone (500 mL) at 50 °C for 6 h under stirring (220 rpm). The defatted spores were then collected by vacuum filtration and air-dried for 12 h. The dried samples were then refluxed (70 °C) in an aqueous 6% (w/v) potassium hydroxide solution (500 mL) under stirring for 6 h. The samples were collected by vacuum filtration and washed with Milli-Q water  (2 × 500 mL) before resuming alkaline lysis for another 6 h using a 6% (w/v) potassium hydroxide solution (500 mL). After 12 h of alkaline lysis, the SDMCs were collected by centrifugation at 4,500 rpm and washed with hot Milli-Q water . After each wash, the suspension was collected by vacuum filtration. The SDMCs were then washed with hot ethanol  and dried overnight under fume hood. The resultant SDMCs were subjected to acidolysis by suspension in 85% (v/v) phosphoric acid (500 mL) and stirring under gentle reflux at 70 °C for 5 h. After acidolysis, the SDMCs were collected and washed sequentially in hot water , hot acetone , hot 2 M hydrochloric acid , hot 2 M sodium hydroxide , hot water , hot acetone  and then hot ethanol . The resulting SDMCs were finally collected by vacuum filtration. The washed SDMCs were transferred to a clean glass dish and air-dried for 12 h. Drying was completed in a vacuum oven  at 60 °C for 8 h, and the dried SDMCs were then stored in a dry cabinet.Briefly, natural Similarly, a defatting process was performed by refluxing camellia bee pollen (250 g) in hot acetone  with magnetic stirring . The acetone was removed by vacuum filtration, and 1 L of warm water was added to the pollen under stirring. The resulting mixture was passed through a 150 µm nylon mesh  to remove any contaminants. Water was removed from the suspension via vacuum filtration. This process was then repeated for another wash cycle.The purified pollen was refluxed again in acetone , isolated by vacuum filtration, transferred to a glass dish, and air-dried under fume hood. The dry pollen powder (20 g) was resuspended in diethyl ether (250 mL) under stirring  at room temperature. The process was repeated for another wash cycle using fresh diethyl ether. For the final wash, the pollen was added to diethyl ether (500 mL) and stirred (300 rpm) at room temperature overnight. The defatted pollen was isolated by vacuum filtration and left to dry under fume hood.The defatted pollen (6 g) was refluxed in 85% (v/v) phosphoric acid (60 mL) at 70 °C for 5 h (220 rpm). The resulting SDMCs were filtered and washed sequentially with 50 mL portions of water (five times), acetone (twice), 2 M hydrochloric acid (once), 2 M sodium hydroxide (once), water (five times), acetone (once), ethanol (twice) and water (once). The samples were dried under fume hood overnight and then in a vacuum oven . The dried SDMCs were stored in a dry cabinet at room temperature until further characterization.Natural cattail pollen grains (10 g) were defatted by refluxing in acetone (100 mL) at 45 °C with magnetic stirring . The pollen grains were then obtained by vacuum filtration and washed with acetone (50 mL). The defatted pollen grains were then dried under fume hood at room temperature for 12 h.To isolate the SDMCs via acidolysis, the defatted pollen grains (2 g) were placed in a poly(tetrafluoroethylene) round-bottom flask containing 85% (v/v) phosphoric acid (15 mL) and refluxed at 70 °C (water bath) for 2.5 h under gentle magnetic stirring (180–200 rpm). After 2.5 h, the flask was removed from reflux and its contents were allowed to cool down to room temperature. The suspension was then diluted with deionized water (150 mL) and vacuum-filtered. The SDMCs were collected in a clean 250 mL beaker and washed with 150 mL of warm water. The warm water wash was repeated five times with vacuum filtration until the pH of the washings reached approximately 6 or 7. The resulting SDMCs were collected in a clean 250 mL beaker, and washing steps were conducted by bathing the capsules sequentially in 100 mL portions of hot acetone (twice), hot 2 M hydrochloric acid (once), hot 2 M sodium hydroxide (once), hot water (five times), hot acetone (once), hot ethanol (twice) and finally hot water (three times). During each wash, the SDMCs were stirred in a beaker to produce a homogenous mixture and to prevent the capsules from aggregating. The solvent was then removed by vacuum filtration. The final washed capsules were then transferred to a clean glass dish and air-dried at room temperature for 12 h.Defatted dandelion pollens (2 g) were mixed with 85% (v/v) phosphoric acid (15 mL) in a 50 mL single-neck flask fitted with a glass condenser and refluxed at 70 °C for 5 h under magnetic stirring (220 rpm). After acidolysis, the SDMCs were collected by vacuum filtration and washed sequentially with 100 mL portions of hot water , hot acetone , hot 2 M hydrochloric acid , hot 2 M sodium hydroxide , hot water , hot acetone , hot ethanol , and hot water . The final product was collected by vacuum filtration, and the washed SDMCs were then transferred to a clean glass dishand air-dried under fume hood overnight. The drying process was continued in a vacuum oven at 60 °C under 1 mbar vacuum conditions for 4 h. Finally, the dried SDMCs were stored in a dry cabinet at room temperature for further characterization.39. For SGFs containing an enzyme, 0.2 wt.% NaCl and 0.32 wt.% pepsin were dissolved in deionized water, and the pH was adjusted to 2.0 by adding 0.2 M HCl to the solution. For SIFs containing an enzyme, 0.68 wt.% monobasic potassium phosphate and 1 wt.% pancreatin were dissolved in deionized water, and the pH was adjusted to 7.1 using 0.2 M NaOH and, in the case of overshoot,0.2 M HCl.The conditions of physiological digestion areas described in the literatureThe SDMCs were divided into three groups that were subjected to different degradation conditions: SGF treatment, SIF treatment and a control group in which water was used as the incubation medium. For SGF and SIF treatment, the SDMCs were segmented into two 40 mg batches, each of which was loaded into a 2 mL Eppendorf tube containing 0.6 mL of SGF or SIF, mixed thoroughly by vortexing for 1 min, and incubated at 37 °C in an orbital shaker incubator  with circular shaking at 220 rpm. For the control group, the SDMCs (40 mg) were incubated in 0.6 mL of deionized water for 24 h under the same incubation conditions. These three batches of SDMCs were then sampled at set durations , isolated by centrifugation at 7,000 rpm for 15 min, washed with SGF or SIF (1 mL) for five times and deionized water (1 mL) for five times and lyophilized.−1. Imaging was performed at a flow rate of 0.1 mL min−1 and a camera rate of 14 frames s−1. At least 10,000 particles were counted for each measurement, and three separate measurements were performed per sample. One thousand well-focused SDMCs were selected by edge gradient ordering and manual processing for the representative images.DIPA was performed using a FlowCAM VS  equipped with a 200 μm flow cell (FC-200) and a 20x magnification lens . After 0.5 mL of prerun, untreated, SGF-treated and SIF-treated SDMCs samples were primed manually into the flow cell at 2 mg mL−1with 16 scans per measurement and 6 replicate measurements per sample.FTIR measurement was performed with a PerkinElmer Spectrum  equipped with a diamond cell attenuated total reflection accessory. Reflectance infrared (IR) spectra were collected in the midinfrared region of 4,000–650 cmx refers to the absorbance value, Background spectra were collected before sample analysis and automatically subtracted from each measurement. Baseline correction was carried out using Spectrum 10 software (PerkinElmer). After baseline correction, each spectrum was standardized as previously reported. Briefly, Untreated and SGF/SIF-treated SDMCs samples were frozen and lyophilized overnight in a freeze dryer  under 0.008 mbar vacuum. A small number of samples were immobilized on a sample holder with carbon tape and sputter-coated with gold to a thickness of 20 nm  using a JFC-1600 Auto Fine Coater  to reduce charging effects during SEM imaging. FE-SEM images were taken with a JSM-7600F Schottky microscope (JEOL) at an acceleration voltage of 5.00 kV. Visual inspection of more than 50 randomly chosen SDMCs particles from all the untreated and SGF/SIF-treated SDMCs samples was performed at different magnifications  to assess any morphological changes in the samples.In order to investigate the morphological nature of the SDMCs, we used DIPA to characterize the four SDMCs species Fig. . The SDM40. Camellia SDMCs are triaperturate, with three cleavage planes 35. The resultant functional group peaks for the SDMCs were assigned according to the literature49 and are summarized in Table −1 is observed for all four species of SDMCs, which is an attribution of UV sensitive aromatic rings in spropollenin35. Several peaks attributed to functionalized carbohydrates  were observed between 1,200 and 900 cm−1, with the peak shapes varying between the different species. A strong peak at 1,745 cm−1, representing lipids, is observed only for the lycopodium SDMCs. Overall, as expected, the spectral differences between the four species of SDMCs, especially between 800 and 1,800 cm−1, were attributed to their different compositions.We performed a comprehensive analysis of the FTIR spectra collected for the untreated and incubated SDMCs in order to identify any chemical changes and possible pathways of degradation Fig. . To remo−1 peak in camellia SDMCs, which was absent after 1 h of incubation in SGF. The absorbance difference spectra, as presented in Fig. −1 range were seen to increase, likely due to the formation of new bonds, such as C–O bond. Similar results regarding the formation of new C–O bonds during the acetolysis of sporopollenin have been reported37. Cattail SDMCs appear to be the most stable and show the least fluctuation in their FTIR absorbance difference spectra. The hydroxyl groups in the lycopodium SDMCs were relatively stable in both SGF and SIF, whereas the other peaks presented a decreasing trend. These results demonstrate that the chemical signatures of SDMCs are altered to various degrees during incubation in gastrointestinal fluids, most likely owing to their species-dependent chemical composition.After incubation treatment, no significant changes in peak shape or number were observed, except for the 1,516 cm−1 peak was set as the internal standard for camellia, 989 cm−1 for cattail, 1,378 cm−1 for dandelion and 994 cm−1 for lycopodium. The peak heights for C = O, C = O, C–H, C–O–C and O–H groups were then standardized relative to the corresponding internal standard. The peak ratios were normalized relative to the untreated SDMCs, and a heat map was generated to better compare the chemical stability of all four SDMCs  that explain the most variable parameters of the data sets. This method has proved useful in the interpretation of FTIR spectra, which show band diversity and complication depending on the source of the sample53. In this work, standardized FTIR data sets, which consist of the wavenumber and the corresponding absorbance, were used to perform PCA. The first PC describes as much of the variability (variation of absorbances) in the data as possible, the second PC, orthogonal to the first, accounts for as much of the remaining variability as possible, and so forth53. We observed that the first two components account for more than 60% of the total variance for each species.Finally, PCA of the FTIR spectra was performed to observe data clustering for the untreated, SGF-treated and SIF-treated samples Fig. . PCA is −1 and 1,516 cm−1 peaks , the morphology of these SDMCs  remained consistent. As surface erosion leads to the loss of surface material, while bulk erosion results in rapid internal degradationIn this study, the degradation of SDMCs obtained from four different species of plants  in simulated human gastrointestinal fluids was studied. Physical characterization of the SDMCs was performed using FE-SEM and DIPA, and their chemical compositions were studied using FTIR spectroscopy, and the resulting data were analyzed using PCA. The four species of SDMCs showed various degrees of chemical degradation; camellia SDMCs were observed to undergo the most significant degradation upon incubation in SGF and SIF, whereas dandelion and lycopodium SDMCs appeared to remain stable. The analysis of our results indicated that bulk erosion is the main mechanism for the degradation of SDMCs in SGF and SIF. Overall, we have demonstrated that the degradation of different SDMCs occurs in a species-dependent manner in response to physiological conditions. We believe that these findings provide insights and understanding that will aid future biological applications of SDMCs.Table S1


To investigate the mortality, operation time, cystectomy time, and complications of anterior approach laparoscopic radical cystectomy (ALRC) in Asian males in comparison with posterior approach laparoscopic radical cystectomy (PLRC).One hundred forty-seven male patients with bladder cancer (cT2-3NxM0) in our hospital from May 2011 to January 2018 having undergone laparoscopic radical cystectomy were studied, including 68 patients in PLRC group and 79 patients in ALRC group. Baseline patient characteristics, operative and postoperative characteristics, and postoperative complications were retrospectively collected and analyzed between the two groups.p > 0.05). Compared with PLRC group, ALRC group required similar operation time (317.3 ± 40.9 vs 321.9 ± 37.5) and cystectomy time (64.8 ± 8.7 vs 65.6 ± 14.0). The ALRC group required less cystectomy time (67.8 ± 10.1 vs 77.4 ± 14.9) when patients’ BMI > 24 or patients had large total tumor and blood clot volume (> 160 cm3). Also, estimated blood loss (EBL) of ALRC group was significantly less than that of PLRC group (477.8 ± 97.4 vs 550.4 ± 99.9). There existed no significant differences between the PLRC and ALRC groups in postoperative characteristics and complications.Patients in these two groups exhibited similar baseline characteristics (3.This study revealed that ALRC required less cystectomy time for patients with higher BMI and larger tumor, suggesting less blood loss and similar perioperative complications. ALRC is recommend for male patients, of which BMI > 24 or total tumor and blood clot volume > 160 cm Bladder cancer (BCa), characterized by high risk of recurrence and mortality, is a serious health risk worldwide, with an estimated 430,000 new cases in 2012 [Radical cystectomy (RC) is the standard management of non-metastatic, muscle-invasive BCa . Though The LRC techniques for male fat patients or male patients with larger tumor and blood clot have been rarely studied. To make better laparoscopic visualization and bigger working space, posterior approach laparoscopic radical cystectomy (PLRC) was modified in 2011, and bladder suspension was performed during the laparoscopic radical cystectomy. To compare PLRC with anterior approach laparoscopic radical cystectomy (ALRC), 147 male patients who underwent PLRC or ALRC in our hospital from May 2011 to January 2018 in this study were retrospectively analyzed, and our experience of ALRC was reported.2-3NXM0) in line with TMN classification of the Union for International Cancer Control [This study was approved by the ethics board of Xiangya Hospital, Central South University, in Changsha, China. All the participants were informed that their clinical information may be applied in later clinical study when they entered hospital. Also, their written informed consents were given. Between May 2011 and January 2018, there were 157 male patients in our hospital, with high-grade bladder urothelial carcinoma, of which 72 and 85 patients are in the PLRC and ALRC group respectively; we failed to follow up 4/72 patients in PLRC group and 6/85 patients in ALRC group. Thus, these patients were excluded, and the medical records of the 147 patients retrospectively were collected and analyzed from our bladder cancer database. We made the technique rotation from month to month. All patients have high-grade bladder urothelial carcinoma  physical status classification system. Each patient underwent preoperative examination including routine laboratory test, echocardiography, chest radiography, lung function test, computerized tomography, or magnetic resonance imaging of urinary system. Common comorbidities were recorded, including diabetes mellitus, coronary artery disease, hypertension, chronic obstructive pulmonary disease, and other chronic diseases. Patients began semi-liquid diet 48 h before surgery, liquid diet 24 h before surgery, and underwent bowel preparation with enema and oral sodium phosphates solution 12 h before surgery. All patients got broad-spectrum systemic antibiotic intravenously during the induction of anesthesia. Same laparoscopic instruments and devices from KARL STORZ were used in both groups. All the operations and perioperative management were performed by the same experienced surgical team. Three surgeons  performed the operations. MC performed 30 and 35, LQ performed 22 and 24, and XZ performed 16 and 20 in PLRC and ALRC respectively. All the surgeons are very experienced and perform more than 25 cases of laparoscopic radical cystectomy annually. Follow-up studies were conducted with ultrasonography and CT scan at 3 and 9 months postoperatively.The baseline patient characteristics included age, BMI, hemoglobin (Hb), total tumor and blot clot volume, comorbid conditions, and ASA score. Total tumor and blot clot volume were measured with CT scan. Operative characteristics included operation time, cystectomy time, estimated blood loss (EBL), and transfusion needed, while postoperative characteristics included Hb, time to liquid intake, time to exsufflation, and hospital stay after surgery. Operation time was defined as anesthesia from the beginning to the end, and cystectomy time was defined as procedure from starting mobilization to bladder removed completely. Intraoperative complication includes rectal injury and external iliac vein injury. Complications that occurred within 90 days after surgery were considered early complications, which included ileus, deep vein thrombosis, pyelonephritis, infection, obturator nerve paresis, and wound dehiscence . The comt test were performed to compare categorical and parametric data respectively between the two groups. Parametric data is denoted as mean ± SD, and categorical data is presented as frequency (%). Linear regression analysis was conducted to assess the relationship between cystectomy time and total tumor and blood clot volume. Significant differences were considered when p < 0.05. Statistical analysis was conducted with the Statistical Package for Social Scientists, version 22.0 (IBM Inc).Chi-square test and Student’s The basic laparoscopic radical cystectomy procedures were performed according to Campbell-Walsh Urology , A five-The posterior approach laparoscopic radical cystectomy is a conventional LRC, the posterior plane was developed first, and the whole operation procedure was performed according to Campbell-Walsh Urology . The antIn general, 147 patients were recruited here, including 68 patients who underwent PLRC and 79 patients proceeded with ALRC. Patients in the PLRC group and ALRC group had comparable baseline characteristics, including age, BMI, Hb, total tumor and blood clot volume, comorbid conditions, and ASA score  and ALRC , cystectomy time were correlated significantly positively with total tumor and blood clot volume. Both PLRC and ALRC required similar cystectomy time when total tumor and blood clot volume was about 160 cm3. Compared with ALRC, PLRC required less cystectomy time and operation time when total tumor and blood clot volume was less than 160 cm3, and PLRC required more cystectomy time and operation time when total tumor and blood clot volume was larger than 160 cm3  and cystectomy time (64.8 ± 8.7 vs 65.6 ± 14.0) were similar between the two groups. When BMI was less than 18.5, the PLRC group took less cystectomy time compared with the ALRC (52.5 ± 6.5 vs 60.3 ± 6.2). When BMI was ranged from 18.5 to 24, cystectomy time was similar between the two groups. It took longer cystectomy time for the PLRC group when BMI was greater than 24 (77.4 ± 14.9 vs 67.8 ± 10.1) , the ALRC group required less time for cystectomy when patients’ BMI was greater than 24 (67.8 ± 10.1 vs 77.4 ± 14.9). Also, when total tumor and blood clot volume was larger than 160 cmLRC should be limited to experienced and skilled laparoscopic urologists. The working space in the pelvis and proper laparoscopic visualization are vital for LRC. The bladder lies deeply in the pelvis with a limited space. For female patients, the operative field may be fully revealed by adjusting the surgical posture to a hyperextension lithotomy position, while it remains hard to achieve good better visualization for male patients. In the meantime, there was no effective method reported in the literature to achieve better visualization except the traction of the assistants’ devices. Here, a safe and effective alternative technique was reported in detail to achieve better visualization in laparoscopic cystectomy for male patients.To sum up, the technique of bladder suspension applied in ALRC is simple and effective to provide better visualization and larger working space. Compared with PLRC, it takes less time of laparoscopic cystectomy for patients with greater BMI or larger total tumor and blood clot volume, and patients also have less blood loss. The technique of bladder suspension is verified to be suitable for Asian male BCa patients, especially those with greater BMI or larger tumor and blood clot within the bladder.