Chronic hyperglycaemia in type 1 diabetes affects the structure and functioning of the brain, but the impact of recurrent hypoglycaemia is unclear. Changes in the neurochemical profile have been linked to loss of neuronal function. We therefore aimed to investigate the impact of type 1 diabetes and burden of hypoglycaemia on brain metabolite levels, in which we assumed the burden to be high in individuals with impaired awareness of hypoglycaemia (IAH) and low in those with normal awareness of hypoglycaemia (NAH).1H magnetic resonance spectroscopy data, measured under hyperinsulinaemic–euglycaemic conditions.We investigated 13 non-diabetic control participants, 18 individuals with type 1 diabetes and NAH and 13 individuals with type 1 diabetes and IAH. Brain metabolite levels were determined by analysing previously obtained p = 0.013) and with IAH , compared with control participants. Cerebral glutamate levels correlated with HbA1c levels  and correlated inversely  with the age at diagnosis of diabetes. Other metabolite levels did not differ between groups, apart from an increase in aspartate in IAH.Brain glutamate levels were higher in participants with diabetes, both with NAH (+15%, In conclusion, brain glutamate levels are elevated in people with type 1 diabetes and correlate with glycaemic control and age of disease diagnosis, but not with burden of hypoglycaemia as reflected by IAH. This suggests a potential role for glutamate as an early marker of hyperglycaemia-induced cerebral complications of type 1 diabetes.ClinicalTrials.gov NCT03286816; NCT02146404; NCT02308293 Type 1 diabetes has a negative effect on the structure and functioning of the brain –4. Struc1H MR spectroscopy (MRS) is a suitable tool for the non-invasive examination of a range of neuro-metabolites in humans ) and suboptimal glycaemic control . Main exclusion criteria were: MRI contraindications, the use of drugs (other than insulin) that may alter glucose metabolism, HbA1c levels >75 mmol/mol (9.0%) and presence of microvascular complications, except for background retinopathy.The cohort consisted of 13 individuals without diabetes, 18 with type 1 diabetes and NAH, and 13 with type 1 diabetes and IAH. Participants with type 1 diabetes were initially classified as NAH or IAH using the Dutch modified version of the Cox questionnaire , 21, andn = 32) or MAGNETOM Prisma-fit (n = 12), Siemens, Erlangen, Germany), using a body coil for excitation and a 12-channel receive-only head coil. A T1-weighted anatomical image  was used for MRS voxel positioning and for determination of the amount of grey matter, white matter and cerebrospinal fluid (CSF) in the MRS voxel.MRS measurements were performed on a 3T MR system (Tim MAGNETOM Trio (1H MRS data were acquired from a single voxel (22.5–25.0 cm3) in the periventricular region of the brain. A semi-LASER spectroscopy sequence , those with IAH and optimal glycaemic control  and those with IAH and suboptimal glycaemic control .Linear regression analysis was performed between glutamate levels and HbAp value less than 0.05 was considered statistically significant. No correction for multiple comparisons was performed.Data are presented as mean ± SD. All statistical analyses were performed in IBM SPSS Statistics 22 . A 1c levels in the patient groups (Table 1c levels) between the subgroups of participants subdivided according to glycaemic control (data not shown). Plasma glucose levels were well in the euglycaemic range during the acquisition of the MRS data, with no differences between groups .The participants were matched for age, sex and BMI, and for diabetes duration and HbA1H MR spectra were of good quality  and in those with IAH , when compared with non-diabetic control participants. Glutamate levels did not differ between the two patient subgroups (p = 1.0). Furthermore, we observed higher aspartate levels in type 1 diabetes and IAH  compared with non-diabetic control participants. All other metabolite levels, as well as the level of macromolecules, were similar across groups . However, an increased flux through the MAS implies opposite changes in glutamate and aspartate levels , which i13C MRS and metabolic modelling. Brain glutamate levels are typically used as an input in such models and often it is assumed that glutamate levels are constant (e.g. no change upon hypoglycaemia) and not different between different groups of individuals  [Our finding of increased glutamate levels in the brain of individuals with type 1 diabetes is also important in the context of studies on brain metabolism with iabetes) , 39. The1c levels below 9.0% and were relatively young. Future research should focus on the effect of more poorly controlled and long-standing diabetes.The present study has some limitations. MRS data were acquired from a large single voxel, which precludes any analysis of the regional dependency of the described alterations. Furthermore, we performed an explorative analysis on multiple metabolites in a rather limited sample size. As a consequence of clamp conditions, insulin levels were elevated albeit within the physiological range. We are, however, unaware of modulating effects of insulin on brain glutamate levels. Furthermore, we used the presence of IAH as proxy for recurrent hypoglycaemia, since habituation to frequent hypoglycaemia underlies the pathogenesis of IAH. We acknowledge that IAH is not an on/off phenomenon, so an impact of hypoglycaemia, which also occurs in individuals with NAH, cannot be fully excluded. Also, we did not actually measure burden of hypoglycaemia, but based on the pathogenesis of IAH we assumed this burden to be greater in people with IAH than in those with NAH. Finally, inherent to the inclusion criteria, the participants with type 1 diabetes all had HbAIn conclusion, we show that there are alterations in neuro-metabolites in individuals with type 1 diabetes. Cerebral glutamate levels are higher in type 1 diabetes and correlate with known risk factors for cerebral decline, suggesting a potential role for glutamate as an early indicator of cerebral complications of type 1 diabetes. The presence of IAH, which usually results from recurrent hypoglycaemia, has only limited additional impact on the neurochemical profile of individuals with type 1 diabetes.


Sargassum horneri) biomass, called ‘golden tides’, has frequently drifted and accumulated along the southern coastlines of Korea, causing devastating impacts on the local economy and coastal ecosystems. In this study, based on combined analyses of mitochondrial DNA cox3 gene and seven microsatellites, we investigated the genetic makeup of the floating S. horneri populations (N = 14) in comparison to Korean benthic populations (N = 5), and tracked their genetic sources. Given a shared mtDNA haplotype and oceanic circulation systems, the floating populations may have been originated from the southeastern coast of China . Population structure analyses with microsatellites revealed two distinct genetic clusters, each comprising floating and benthic populations. High levels of inter-population differentiation were detected within Korean benthic samples. The floating populations from the same periods during a 2015–2018 year were genetically more different from one another than those from different periods. These results suggest that the floating populations might be of multiple genetic sources within geographic origin(s). This study will inform management efforts including the development of “S. horneri blooming forecasting system”, which will assist in mitigating ecological and economic damages on the Korean coastal ecosystems in the future.In recent years, drifting and inundating brown seaweed ( The green seaweeds, Ulva including formerly known as genus Enteromorpha and the brown macroalgae, the genus Sargassum are especially notorious for the formation and inundation of drifting seaweed biomass along the coastlines globally2. Shortly after being accumulated along the coastlines forming like carpets, these seaweeds begin stinking by releasing toxic hydrogen sulphide (H2S), rendering devastating effects on the coastal ecosystems4. The physiologically atypical capability of sustaining their lives as an unattached (drifting) form of these algae enables them to easily build up a large amount of biomass, colonize new space, and form the massive seaweed tides1.Recurrent outbreaks of drifting seaweed masses, called ‘seaweed tides’, are a nuisance phenomenon worldwide, as they pose a serious threat to local biodiversity in coastal ecosystems and also cause severe damage to the local economy by disturbing tourism, aquaculture and fisheriesSargassum spp., called “golden tides”2 accumulates nearshore coastal waters usually during spring to summer, which causes detrimental effects on aquaculture and fisheries and also the whole coastal ecosystems in many parts of the world including the Gulf of Mexico, Caribbean, West Africa, eastern Pacific coast, China coast and the southwestern coast of Korea9. This phenomenon of the golden tides has been intensified dramatically in frequency, range, and magnitude over recent years perhaps due to growing anthropogenic pressure, such as climate change, nutrient loadings  and inadvertent algal species transport by ship movements11.Drifting biomass of Sargassum horneri is known to be the major golden-tide seaweed in the northwestern Pacific coasts including China, Japan and Korea12 as well as the northeastern Pacific coasts13. More recently, S. horneri that bloomed in the eastern coast of China has frequently drifted in a large biomass with oceanic currents and accumulated along the southern coast of Korea including Jeju Island, off the southernmost region of the mainland, and also the South and West Sea since 20155  form kelp forests . This species is dioecious with ellipsoidal pneumatocysts and has an annual life cycle with sexual reproduction using reproductive receptacles15. However, the reproductive mode of S. horneri in a floating state has never been examined, although an earlier study of floating Sargassum species in the Sargasso Sea suggested vegetative reproduction for their entire lives16.Despite its detrimental effects as the golden tide seaweed, S. sts Fig.  and playS. horneri populations in Korean waters are presumed to originate from certain areas in the east coast of China9. The Korean coast has a geological position that is primarily influenced by the Kuroshio Current as well as the Yellow Sea Warm Current and the Tsushima Warm Current that merge into the Kuroshio Current17. Because oceanic current systems are intricate and dynamic, it is, however, difficult to uncover the precise geographic origins of floating populations solely based on satellite images with the ocean currents.Given the direction of oceanic currents (e.g. the Kuroshio Current) according to satellite image data, floating 18. Several phylogeographic and population genetic studies have been performed to identify species/strains and source populations and pathways of colonization or to understand the genetic diversity and population structure dynamics of several nonindigenous seaweed species, such as Asparagopsis spp.19 and Sargassum natans20 and also our study species, S. horneri21. A recent genetic survey of floating and benthic populations of S. horneri primarily focused on the east coast of China (the Yellow Sea) and sample sizes of Korean samples used for their study are limited (N = 1 for each of the five samples)21. A more recent study investigated the genetic composition of only floating samples of S. horneri exclusively from China’s east coast22. Hence, the phylogeographic/population structure and genetic diversity of drifting populations of S. horneri in Korean waters remain largely unknown. Also, the genetic structure of benthic populations of S. horneri on the Korean coast has not yet been studied.Genetic analysis may provide a useful tool for identifying strains, and tracking geographic origins of nonindigenous populationscox3), we aimed to trace the geographic or genetic origins of 14 floating S. horneri populations to the Korean coasts including Jeju Island, the South Sea and the West Sea, and also in the East China Sea (ECS). We also analysed and compared the genetic structure between floating (N = 14) and benthic populations (N = 5) in Korean waters, based on seven nuclear microsatellite markers. Using floating populations collected at different time periods during a 2015–2018 year, we further tested whether there were changes in their genetic compositions over time. The results of our study will provide important insights into the golden-tide seaweeds in Korean waters and inform management efforts including the development of “S. horneri blooming forecasting system” that is now underway by the Korean government, which will help to prevent or mitigate ecological and economic damages on the Korean coastal ecosystems in the future.In this study, by performing a phylogeographic analysis of mitochondrial DNA (mtDNA) cytochrome c oxidase III  from 18 different localities along the Korean coasts including the South Sea, the West Sea and the East Sea as well as Jeju Island, which is off the southernmost region of the mainland, and also in the ECS from August 2015 to January 2018 (Table N = 14) included KF15, KF16-1, KF16-2, KF17, CF17, CF17a, KF17a-1, KF17a-2, KF17a-3, KF17a-4, KF17a-5, KF17a-6, KF17b-1 and KF17b-2. Of those, CF17 and CF17a were comprised of drifting populations from the ECS, approximately 250 km off the Korean Peninsula, and sampled twice from the same areas at different time periods in 2017 to test for the temporal genetic structure (Table N = 5) included K1 and K2 from the East Sea, K3 and K4 from Jeju Island, and K5 from the South Sea . Powdered samples were stored in a 1.5 ml microcentrifuge tube with silica gel and stored at −20 °C until genetic analysis.cox3 gene, previously shown to possess relatively higher polymorphism than chloroplast DNA markers23, was amplified for 230 samples using the published primers CAF4A and CAR4A24. Polymerase chain reaction (PCR) amplification was carried out in a reaction volume of 15 μl containing 1 × PCR buffer, 25 μM of each dNTP , 0.6 μM of each of the forward and reverse primers, 0.2 units of Taq DNA polymerase  and approximately 5−10 ng of genomic DNA. RCR thermal cycling conditions comprised an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 50 °C for 50 s and extension at 68 °C for 60 s, and a final extension at 68 °C for 10 min. PCR products were checked on 2% agarose gels stained with Redsafe (iNtRON). The amplified mtDNA were purified enzymatically with Exonuclease I  and Shrimp Alkaline Phosphatase (New England BioLabs). The purified mtDNA fragments were subjected to direct sequencing only in the reverse direction using the same reverse primers as in the PCR and the BigDye Terminator 3.1 Cycle Sequencing Ready Reaction Kit in an ABI 3730xl automated DNA sequencer . The DNA sequences were edited using CHROMAS Lite v2.1.1 computer software and aligned with Clustal W25 implemented in BioEdit v7.2.526, and finally verified manually.Genomic DNA was extracted from pulverized leaf samples using i-genomic Plant DNA Extraction Mini Kit . A partial fragment (469 bp) of mtDNA 27. Each of the forward primers was labelled with a fluorescent dye . PCR reactions were conducted as described for the mtDNA cox3 gene. PCR cycling conditions were carried out as suggested in a previous study27. The PCR products were electrophoresed on an ABI 3730xl automated DNA sequencer (Applied Biosystems). Fragment sizes were determined with the ROX 500 bp size standard (ABI) using GENEMAPPER software v5.0 (Applied Biosystems).Seven polymorphic nuclear microsatellite loci were amplified with the published primers including SHORN18, SHORN26, SHORN27, SHORN30, SHORN31, SHORN34 and SHORN 41S. horneri, partial sequences of mtDNA cox3  representing the northwestern Pacific lineages including Chinese and Japanese clades were retrieved from GenBank17. Only 45 previous haplotype sequences were used for the final analysis, as the 51 haplotypes were merged into 45 haplotypes due to adjustments to a shorter length of 469 bp. The best nucleotide substitution model was tested using initial searches according to the AICc criterion with jModelTest v2.1.728, which selected the Jukes-Cantor (JC) model. The phylogenetic tree was then reconstructed with maximum likelihood (ML) method using as implemented in Mega v7.029. Sequences of S. muticum as outgroup were retrieved from GenBank (Accession Nos.: AB430582.1) and statistical support was estimated by 1000 bootstrap replicates.To trace original genetic sources of floating populations to the Korean coast, and also to determine phylogeographic relationships between mtDNA haplotypes of DA distances among the 19 samples were calculated using POPTREE230.Phylogenetic analysis was also conducted with a neighbour joining (NJ) approach, based on microsatellite allelic variation. S. horneri, the mean number of alleles per locus (NA), observed (HO) and expected (HE) heterozygosity, and allelic richness (AR) corrected for unbalanced sample sizes among the samples were calculated using GENEPOP v4.331 and FSTAT v2.9.3.232. The AR values were estimated after excluding the samples of KF17a-1, KF17a-5, KF17b-1 and K1 because of insufficient sample sizes (N < 10). A Mann-Whitney U test was performed to examine whether there was a significant difference in the level of AR between floating (N = 11) and benthic (N = 4) populations of S. horneri. The four samples with limited sample sizes were not included for downstream analyses, with the exception of AMOVA, STRUCTURE, FCA and phylogenetic analyses (see below). Multilocus tests for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) tests for genotypes among pairs of the seven loci were also performed in GENEPOP. The 95% significance levels for every exact test for both HWE and LD were corrected using a Bonferroni correction. We also tested for the presence of null alleles across the seven loci using MICROCHECKER v2.2.333 with 1000 randomization at the 95% confidence level.To investigate differences in levels of microsatellite diversity among the 19 samples of S. horneri, calculation of pair-wise FST estimates among the 15 populations as well as exact tests for population differentiation were performed using GENEPOP. The 95% significance levels for the pairwise comparisons were adjusted using a Bonferroni correction. Hierarchical analysis of molecular variance (AMOVA) was performed in ARLEQUIN v3.5.134 to assess temporal genetic structure among floating samples from the different sampling periods, based on microsatellites. The temporal AMOVA was performed by classifying the 14 floating populations into five different groups according to sampling periods .To assess the degree of genetic differentiation between and within floating and benthic populations of 35 under a model of admixed ancestry among populations and correlated allele frequencies with no a priori information on the geographic origins of the samples. STRUCTURE calculates a likelihood score when the data are forced into a given number of genetic clusters (K) = 1–19. We applied 10 iterations, with 10,000 burn-in steps followed by 100,000 Markov Chain Monte Carlo (MCMC) generations. STRUCTURE analyses were also carried out separately for floating populations and also for benthic populations at K = 1–14 and K = 1–5, respectively. The most likely number of genetic clusters  was determined by using the ΔK method35 implemented in the web-based tool STRUCTURE HARVERSTER (http://taylor0.biology.ucla.edu/structureHarvester/)36, based on the rate of change in the probability of data between successive K values. In addition, factorial correspondence analysis (FCA) based on genetic relationships between individuals with multi-locus genotypes was conducted using GENETIX v4.05.237.The genetic structure among and within floating and benthic populations was further analysed using an individual-based Bayesian population assignment approach implemented in STRUCTURE v2.3.4cox3 haplotypes from a total of 230 S. horneri individuals that we sequenced. While the 17 populations including 14 floating  and three benthic  populations shared an identical haplotype (Hap 1), the remaining two benthic populations (K1 and K2) from the East Sea shared another unique haplotype (Hap 2)  that was predominantly distributed in the northeastern coast of Japan  that was observed in populations exclusively from Zhoushan, Zhejiang province across nearly entire east coasts of China and also from the western coast of Japan .The estimated frequencies of null alleles at the seven loci ranged from −0.11 (SHORN41) to 0.09 SHORN34), indicating a low probability of null alleles. Microsatellite diversity indices 4, indicaFST estimates for the microsatellites between the 15 populations revealed significant genetic differentiation except eight comparisons, which were always from those within floating populations , but a highly significant genetic structure within temporal groups  .The temporal AMOVA based on microsatellites revealed absence of genetic structure among the five temporal groups (1) Table . These fK = 2) that were comprised of 13 floating (except KF17a-3) and five benthic populations for each separate cluster 17. The Hap 3 is only found in the floating samples of KF17a-3 and KF17a-4 from the South Sea across all the populations examined, which has never been detected in previous studies.Information on geographic or genetic origins (sources) of the floating S. horneri individuals share the Hap 1, which was found only in the Zhoushan, Zhejiang province across the east coasts of China (H7 in their study)17 might suggest that the populations from this area represent geographic or genetic sources of floating populations in Korean waters. This hypothesis is plausible, especially given influential ocean current systems (e.g. the Kuroshio Current) around this region39. Although the Hap 1 is also relatively common along the western coast of Japan, populations from this area are highly unlikely to serve as source populations to the Korean coast, considering the direction of major oceanic currents, such as the Kuroshio Current and the Tsushima Warm Current nearby. Therefore, given the shared distributions of mtDNA cox3 haplotypes between the Zhoushan, Zhejiang province and floating populations in Korea as well as the oceanic circulation systems in this region, the drifting S. horneri biomass in Korean waters may have been originated from this particular region on the southeastern coast of China. A previous study demonstrated that drifting S. horneri rafts of origins of Zhejiang province can disperse up to the eastern ECS under the effect of the Kuroshio Current12. A more recent study provides genetic evidence supporting the hypothesis that floating S. horneri rafts in the Yellow Sea might be of origins of Zhejiang region40. Given those earlier work and satellite data, our findings may not be unexpected, but the results of our study can provide direct (genetic) evidence supporting the hypothesis that floating golden tide seaweeds from the southeastern coast of China actually travel up to the Korean coast with ocean currents5.Our findings that although most Korean benthic samples also possess the same haplotype, the floating cox3 gene has frequently been used as a molecular marker to examine intraspecific polymorphism for S. horneri22, variation in the cox3 is not sufficiently enough to discriminate between benthic and floating populations in our study. Hence, further studies using more polymorphic markers with additional samples from “benthic” populations on the east coast of China, particularly from the Zhoushan region will be required to determine the origins of floating S. horneri biomass more precisely. Currently, we are developing molecular markers that can differentiate between floating and Korean benthic populations by exploring organelle genomes, but it may not be feasible because mt- and cp-DNA genomes of Sargassum species are known to be relatively conservative23. We are also applying recently developed microsatellite markers, which are assumed to be more polymorphic than ones used in this study, to testing whether they will provide a better resolution for elucidating the genetic structure of floating samples41. However, more extensive sampling, covering particularly from China’s east coast is the most critical for determining the origins, movement patterns and population structure of floating S. horneri biomass. For this, collaborative efforts, such as sharing S. horneri (DNA) samples for genetic analysis, with a counterpart of China through establishing a research consortium will help to advance our current knowledge of the golden tides in this region.Although mtDNA cox3 gene did not house enough variation to discriminate between benthic and floating populations, we applied more polymorphic microsatellites as an additional marker-set to assessing the genetic structure between these samples. Although a mtDNA molecule has been useful as a phylogeographic marker for inferring population or evolutionary histories of seaweed species, hyper-variable microsatellite regions may be more appropriate for understanding ‘contemporary’ processes of seaweed populations as they usually possess higher mutation rates17. Differences in the levels of polymorphism between mtDNA and microsatellite markers can also be linked to the pattern of maternal inheritance of mitochondria42. Mitochondrial inheritance is suggested to be maternal in brown algae43, although this has not been confirmed for S. horneri. The results of microsatellites show that floating S. horneri populations are clearly genetically different from benthic populations (except the KF17a-3 population), as suggested by our multifaceted-lines of analyses such as pairwise F-statistics, STRUCTURE, FCA and also NJ phylogeny. When we investigated all populations combined, strong genetic structure was found between floating and benthic samples, as indicated by two genetic clusters determined by STRUCTURE and FCA analyses. Unexpectedly, the floating KF17a-3 population, which was collected from stranded biomass on Porphyra cultivation rafts, shares a gene pool with Korean benthic populations, except for the three individuals that belong genetically to floating populations 44. Also, naturally detached S. horneri thalli due to wave action were sometimes observed in this area . Nevertheless, a further study with benthic samples from those particular sites will be needed to substantiate our hypothesis.Since the ons Fig. . We hypoS. horneri most likely results from limited contemporary gene flow occurring across these geographically disconnected populations42.When benthic and floating populations analysed separately, benthic populations show considerable genetic structure with four genetically unique groups, whereas floating populations had similar genetic structure with a rather, single genetically homogeneous group except for the KF17a-3 population. The Korean benthic populations are genetically more diverse and substantially divergent to one another with their own genetic integrities: one represents the East Sea (K1 and K2), one the south of Jeju Island (K3), one the north of Jeju Island (K4) and one the South Sea (K5). The observed spatial variation in the genetic structure among the benthic populations of S. horneri rafts represent indistinguishable genetic structure, at least in part due to the overall low levels of genetic diversity within floating samples, possibly caused by the vegetative growth during their floating life stage after detached from the benthic substrata16. Although drifting Sargassum species in the Sargasso Sea was shown to be solely vegetatively reproduced16, whether our study species, S. horneri uses the same reproductive strategy in a floating state is unknown. Therefore, the reproductive mode of floating S. horneri will need to be evaluated to test this hypothesis. Since 2011, the Chinese government has run a large-scale transplantation and farming project for the purpose of the construction of S. horneri forests around Zhoushan, Zhejiang province, which span an area of approximately 860,000 square kilometres45. Most seaweed farming exercises with a limited number of parental strains, which ensues the loss of genetic diversity in the entire Zhoushan population of S. horneri.On the other hand, the drifting S. horneri populations due to the temperature gradient of seawater along the latitude46, gene flow between floating and benthic populations might be hampered because of the differences in the timing of maturation (e.g. receptacle formation), given approximately one-to-three degree differences in the latitude between Zhoushan region of China and southern coasts of Korea. Another possible explanation would be that the establishment of southern floating populations on the Korean coast may be hindered because already established Korean benthic populations preoccupy space and resources, having negative impacts on their successful establishment (i.e. priority effect)47. Lastly, we could not simply sample admixed individuals between floating and benthic populations, although they are present. A more detailed analysis of benthic populations from areas where drifting assemblages are frequently observed will allow to validate the presence or absence of ‘introduced’ or ‘admixed’ individuals.While floating populations have continuously been introduced into Korean waters, particularly recently, the observed lack of gene flow between these and Korean benthic populations can be explained by several hypotheses. First, based on previous studies that found differences in the timing of reproduction in 21, we predicted that floating populations from the same periods will be genetically more uniform than from different periods, since they are more likely to be originated from the same origins. However, weak but some significant genetic variation was observed within floating populations particularly from the same periods, suggesting there may be multiple genetic sources being present. These findings imply that drifting populations in Korean waters at different locations from the same periods would consist of genetically dissimilar individual clusters, which may have been originated from populations with genetically different multiple patches within geographic origin(s). Therefore, the most plausible scenario would be that after S. horneri individuals were detached from genetically distinct natural beds in the southeastern coast of China , albeit low genetic diversity apparent, they may undergo a rapid vegetative growth in the ocean during their drifting lives16 and genetically heterogeneous massive seaweeds may then subsequently drift into the Korean coast49. Our temporal AMOVA seems to support this hypothesis that the genetic variation observed among the five temporal groups was considerably lower than within the groups. A recent study of mtDNA marker suggests that the floating golden tide populations in the Yellow Sea comprise at least two genetic lineages or sources22, supporting the hypothesis that multiple genetic sources exist for floating S. horneri rafts in Korean waters in this study.Based on a previous studyFST = 0.08−0.14, P < 0.05; Table FST = 0.05, P < 0.05), suggesting there may be of different genetic sources. Alternatively, assuming that drifting S. horneri individuals could undergo sexual reproduction, the considerable temporal variation can be interpreted as their low effective population sizes (Ne) that are small enough to be subject to genetic drift (‘sweepstakes’ hypothesis50). In other words, the reproductive success of the ECS population of S. horneri may be like a sweepstakes, in which only a few individuals contribute to producing offspring recruit to the whole population, while a majority do not yield offspring at all51.Yet, some genetic variation exists within temporally varying floating samples. For example, the KF17 floating population in Jeju Island in 2017 differs in the genetic structure from previous floating populations during a 2015–2016 year .In conclusion, although precise genetic sources of the floating S. horneri rafts can be removed directly from the ECS prior to their arrival to the Korea coast, if the source areas and the timing of golden tide blooming are ascertained. This idea may not be unrealistic, considering a government-funded project of ‘the development of S. horneri removal system off the ocean’ launched in 2018 in Korea. Second, establishing an international consortium with counterparts of China and possibly Japan for making cooperative efforts, such as sharing information on the origins/timing as well as the magnitude of S. horneri blooming in situ will be effective to forecast a particular golden tide in Korean waters. Third, applying floating S. horneri biomass as potentially valuable industrial resources, such as food, medicated or agricultural fertilizers is also important for developing the effective management2. Lastly, once floating golden tide seaweeds introduced to the Korean coast, developing a molecular-based fast and accurate detection method is important for their rapid removal. For this, constructing a genetic database for Korean benthic populations will be needed. This study, to our knowledge, first provides genetic information on floating and benthic populations of S. horneri in Korea and will inform management efforts including “S. horneri blooming forecasting system”, which helps to protect and mitigate ecological damages on the Korean coastal ecosystems.For management efforts, we suggest some possible controls to avoid or minimize damages on the Korean coast, based on our findings. First, drifting Supplementary Figure S1


To evaluate the intense pulsed light (IPL) therapy with optimal pulse technology  as an adjunct therapy for the prevention of recurrences in moderate to severe blepharokeratoconjunctivitis (BKC).  This open-label nonrandomized clinical trial evaluated 33 patients diagnosed with BKC. Twenty-one patients received four bilateral OPT therapy sessions with Meibomian gland expression (MGX) (treatment group), and 11 patients received MGX alone (controls). This trial was initiated after a four-week pharmacotherapy for BKC in both groups and was scheduled at four-week intervals. Efficacy outcome measures included meibum quality, Meibomian gland (MG) secretion function, eyelid margin signs, corneal fluorescein staining (CFS) score, noninvasive keratography breakup time (NIKBUT), ocular surface disease index (OSDI) score, Schirmer I test (SIT), classification of tear film lipid layer (TFLL), and Meibomian gland dropout (MGDR). Safety outcome measures included visual acuity, intraocular pressure, eye structure damage, and facial skin appearance at each visit. p < 0.05). While these improved in both groups in comparison to baseline, the NIKBUT and upper and lower eyelid MGDRs significantly improved only in the treatment group (p < 0.05). No adverse events occurred in both groups. No BKC recurrences were noted in the treatment group.  Quality of meibum, MG expressibility, eyelid margin signs, and OSDI score showed a statistically significant greater improvement in the treatment group after one to three treatment sessions, compared to controls (ChiCTR-ONN-17013864. IPL is a safe and effective adjuvant treatment for BKC and possibly more effective in reducing eyelid margin inflammation and prevents recurrences than MGX alone. This trial is registered with  Blepharitis is a common subacute or chronic inflammation affecting bilateral eyelid margins' skin and mucosa, eyelash follicles, and other adnexal glands. When this chronic inflammatory disease of the palpebral margin is complicated with secondary conjunctivitis and keratopathy, it is clinically referred to as blepharokeratoconjunctivitis (BKC) . The cliSince BKC and MGD share some common pathological mechanisms, including inflammation, occlusion of Meibomian gland, and new blood vessels sprouting at the eyelid margin, we hypothesized that the IPL may also carry an anti-inflammatory effect in BKC patients. Compared with the original IPL technology, the fifth generation of IPL with optimal pulse technology  has better safety, efficacy, and reproducibility that can eliminate energy peak at the beginning of the pulse, avoid ineffective decline at the end of the pulse, and provide homogeneous “squared off ” energy distribution with continuous contact cooling . We therChiCTR17013864).We conducted an open-label nonrandomized controlled clinical trial, which enrolled moderate to severe BKC adult patients from January 2018 to February 2018 in the Ophthalmology department of the Beijing Tongren Hospital, Capital Medical University. This study was approved by the ethics committee of the Beijing Tongren Hospital, Capital Medical University, and all participants had signed informed consents before treatment was initiated. All the examination procedures were done in accordance with the Declaration of Helsinki and ethics standards as well as specifications of the Chinese clinical trial research studies. The study was enlisted in the clinical trial registry  at each visit.In all recruited patients, the keratoconjunctivitis was first controlled with topical eye drops for one month, and then the OPT combined with Meibomian gland expression (MGX) (treatment group) or MGX therapy alone (controls) were individually suggested. Treatment was initiated by the patient's preference and was repeated at monthly intervals for four consecutive months. The baseline and the four follow-up visits were coded as The patients in this study met all of the following inclusion criteria: (1) age older than eighteen; (2) bilateral disease; 3) documented signs of blepharitis, including eyelid hyperemia, capillary dilation, scales, scabs, ulcers of the eyelash root, and/or morphological changes of the Meibomian glands; (4) having concomitant conjunctival and corneal lesions, namely, conjunctival congestion, papillary hyperplasia  [ documentPatients were excluded from the study if their diagnosis was not consistent with blepharitis-associated keratoconjunctivitis or if they had any of the following conditions: (1) acute inflammation or allergic eye or periocular skin disease; (2) underlying diseases that can be triggered by an exposure to wave lengths between 560 nm and 1200 nm, such as recurrent herpes simplex infections, systemic lupus erythematosus, or porphyria; (3) current pregnancy or lactation; (4) history of radiotherapy or chemotherapy treatment within the first year prior to the study or scheduled radiotherapy or chemotherapy within the two months after the planned OPT treatment.Initial treatment was decided upon the degree of corneal and conjunctival pathology. One drop of 0.1% fluorometholone  was topically administered three or four times a day; one drop of 0.3% gatifloxacin eye gel  was topically administered once or twice daily; and one drop of 0.3% sodium hyaluronate artificial tears  was administered four times a day. The keratitis was reexamined after two weeks of treatment. The dosage of corticosteroid eye drops was then gradually tapered, and the other eye drops were discontinued in all the patients within one month of the resolution of corneal manifestations. Only sodium hyaluronate eye drops were continued two to three times per day thereafter. For patients with facial seborrheic dermatitis or acne rosacea, a dermatologist was consulted to determine the appropriate systemic drug regimen. Patients with rosacea were treated with minocycline hydrochloride capsules 50 mg , twice a day, and with Fusidic cream  twice a day. Patients with seborrheic dermatitis were given oral dantone capsules 0.25 g, three times a day , and selenium disulfide lotion  was used twice a week. Oral medications were stopped before OPT + MGX or MGX was initiated.After one month of topical drug treatment, keratoconjunctivitis was resolved in all subjects and the treatment of OPT/MGX or MGX alone was initiated in the treatment and control groups, respectively. All treatment sessions were performed by a single physician (Y.J.) using M22 OPT technology of Lumenis Medical Laser Co., Ltd. The procedure was performed taking the following measures:(1) Eye Protection. Wet dressings were applied for skin and periocular hair protection. The physician was instructed to wear protective glasses.(2) Intensity Adjustment. An 8 mm × 15 mm optical crystal cooling head was used, and the treatment parameters were tailored upon the patients' skin types [2, optical filter 560 nm, pulse quantity 3, pulse time 3.5 ms, and pulse delay 20 ms. For Fitzpatrick skin type IV, the settings were energy density 12 J/cm2, optical filter 590 nm, pulse quantity 3, pulse time 3.5 ms, and pulse delay 25 ms. Both cheeks were exposed to a test flare, and in the lack of any skin reaction, treatment was initiated 5 minutes later.in types . Based o(3) OPT. Medical ultrasonic couplant  was applied to locate the treatment areas. Eleven points were then marked, including eight points at the lower eyelid margin in two lines from medial to lateral, two more points at the outer canthus, and one more point at the nasal alar. The treatment was done symmetrically on both sides. The coupling gel layer covering the treatment area was about 1-2 mm in thickness. The upper eyelid was not treated directly to avoid a possible light damage to the intraocular structures. The optical crystal directly touched the coupling gel in this area and lightly touched the skin, avoiding any pressure exertion. A pulse was emitted every 1-2 seconds. The coupling agent was removed after two repeated therapy sessions in the treatment area (ent area .(4) MGX. A single 0.5% proparacaine hydrochloride eye drop  was applied to the conjunctival sac. Then, the Yoshitomi Meibomian Gland Compressor  was used to perform the Meibomian gland massage on the upper and lower eyelids. A small amount of tobramycin dexamethasone eye ointment  was administered to the palpebral margins after the double eyelids' massage.(5) Treatment regimen. OPT/MGX or MGX therapy alone was initiated one month after pharmacotherapy was first started and was repeated four times at one-month intervals. Four follow-up visits were scheduled at week 4 (V1), week 8 (V2), week 12 (V3), and week 16 (V4) after the first therapy session to assess the eyelid margin, cornea, and conjunctiva by the same ophthalmologist.All the patients had their baseline eye exam recorded before their initial treatment with OPT/MGX or MGX alone. The examinations were performed at the following sequence: visual acuity, noncontact IOP , slit-lamp examination, and direct ophthalmoscopy examination were performed first, followed by the BKC-related examinations 30 minutes later. These included CFS score, NIKBUT, OSDI, SIT grading, grading of TFLL, and MGDR. The sequence of examinations was kept identical for each follow-up visit, and they were performed by the same single ophthalmologist (F.R).Primary outcome measures of treatment's efficacy included quality of meibum, the expressibility of the Meibomian glands, and changes of eyelid margin. Secondary outcome measures of efficacy were assessed by CFS, NIKBUT, OSDI, SIT, grading of TFLL, and MGDR.Outcome measures were assessed as follows.Meibomian gland evaluator 1000  was located 1-2 mm inferior to the eyelid margin, and the central eight glands of the upper and lower eyelids were gently pressed. The liquid extracted from the Meibomian glands was graded by a classification method described by Bron et al., by which 0 = clear fluid, 1 = cloudy fluid, 2 = cloudy particulate fluid, and 3 = inspissated, toothpaste-like discharge. Each of the central eight glands was separately graded, and a score ranging from 0 to 24 was then given to each eye .The central five glands of the upper and lower eyelids were pressed. The following grading system based on Pflugfelder et al. study was used, by which 0 = all glands were expressible, 1 = 3-4 glands were expressible, 2 = 1-2 glands were expressible, and 3 = no glands were expressible. Score ranged from 0 to 3 points .Evaluation of the eyelid margin status was based on the following five signs: blunt rounding shape of the posterior eyelid margin, irregularity or notching of the eyelid margin, and the presence of trichiasis or distichiasis, anterior blepharitis, vascularity, or telangiectasia of the lid margin. One point was assigned to each clinical sign, and the grade ranged from 0 to 5 points .Using the Fluo Imaging corneal dot stain observation program , the cornea was divided into five regions and graded based on dye distribution on the background of cobalt blue light. Grading ranged from 0 to 15 as follows: 0 = no staining; 1 = 1–5 punctate staining; 2 = 6–15 punctate staining; and 3 = any of the following: ≥16 punctate staining, ≥1 long 1 mm staining sites, any filamentous staining [Using the K5M tf-scan tear film analysis procedure, the quality and the stability of the tear film were evaluated by a noncontact and fully automatic method. The device automatically recorded the time at first tear breakup point and its location, starting measurements 1.5 seconds after the patient's second blink. Values below 10 seconds were considered pathological.The OSDI questionnaire was used to assess the extent of patients' discomfort. The questionnaire included 12 questions . Final gThe Schirmer test I was performed using a filter paper (5 mm × 35 mm Whatmann no. 41) placed inside the lower eyelid. The filtered paper was taken out after five minutes, and the amount of wetting was measured in millimeters. Exam was considered positive if wetting of the paper was 5 mm or less.Using the K5M, the thickness and stability of the lipid layer were evaluated and the TFLL score ranged from one to five as follows: grade 1: gray, uniform stripes; grade 2: grayish white, but with slight stripes change; grade 3: yellow stripes appear; grade 4: a jumble of colored streaks; and grade 5: black dry spots . A gradeUsing the Meibo-Scan of the K5M, the structure of the Meibomian glands was observed by an infrared light source, and the loss of the glands was then scored from one to three as follows: 1 = the loss of Meibomian glands was less than 1/3 of the total area; grade 2 = the loss of Meibomian glands accounted for 1/3 to 2/3 of the total area; and grade 3 = the loss of Meibomian glands accounted for 2/3 or more of the total area .The skin at the treatment site was evaluated for any temporary pigmentary changes, alterations in skin sensation, including tingling, itching, or burning, rashes or blisters, skin edema, signs of an active Herpes Simplex virus infection, or inflammatory hypertension. The ETDRS best-corrected visual acuity and the intraocular pressure were measured before and after treatment. slit-lamp biomicroscopy was performed to rule out any conjunctival, corneal, iridal, or lenticular damage. Any iris depigmentation was documented. The direct ophthalmoscope was then used to perform a dilated fundus exam.p value ≤ 0.05 was considered statistically significant.SPSS 19.0  was used for statistical analysis. One-way repeated measure analysis of variance enabled comparison of data across the various time points, paired analyses allowed comparison of pre- and posttreatment data at individual time points and multifactor variance analysis permitted comparison between the two groups. Data are reported as mean ± SD. Among the 21 adult BKC patients (42 eyes) who consisted the treatment group, there were 13 women (61.9%) and 8 men (38.1%) with a mean age of 42.93 ± 13.25. The 11 adult BKC patients (22 eyes) in the control group consisted of seven women (63.6%) and four men (36.4%) with a mean age of 47.62 ± 14.92.p=0.014 and 0.008, respectively) from the second treatment session and on, while the difference in MG expressibility became evident as early as the first session . Differences remained significant for the entire follow-up period.As shown in p=0.022), and kept significant till the last visit, while in the upper eyelids, a statistically significant difference was noted only at the second visit (p=0.041).Eyelid margin signs improved in both groups of patients. A statistically significant difference between the groups was evident in the lower eyelids, as soon as the second visit (p > 0.5). Nevertheless, during the treatment sessions CFS scores significantly differed from baseline in both groups, while the difference in NIKBUT in comparison to baseline was only observed in the treatment group. Subjective symptoms, as reflected by the OSDI, significantly improved after the first therapy session in both groups, and the treatment group showed more subjective improvement as soon as the third follow-up visit and thereafter (p=0.029 at V3 and p=0.049 at V4). No statistically significant differences were observed among the two groups when comparing the SIT and tear film lipid layer classification, as seen in p=0.044 for upper eyelid and p=0.016 for lower eyelid).As demonstrated in Five patients in the treatment group (23.8%) and three patients in the control group (27.3%) had associated dermatologic diseases. After four OPT sessions, the ocular signs and facial skin lesions showed much improvement, indicating high patient satisfaction, while there was no change in dermatosis in the control group.None of the participants in the treatment group experienced a decrease in best-corrected visual acuity, and intraocular pressures were measured <21 mmHg in all eyes. Only three of the patients (14.3%) reported a burning sensation at the area treated with OPT, but the symptoms resolved after the adjustment of energy parameters without further influencing the treatment. Only 1/21 patients endured hair loss attributable to the proximity of the treatment area to patient's hair line; however, hair regrowth soon pursued on the next visit. No recurrence of BKC was observed in the treatment group, while 2/11 patients (18.2%) in the control group had a documented BKC recurrence at the second or third visit, respectively. No other adverse effects were documented.Blepharokeratoconjunctivitis represents a group of recurrent corneal and conjunctival diseases associated with anterior and posterior blepharitis . PosteriDemodex folliculorum mites and Bacillus oleronius bacterium which are potential mediators of blepharitis and MGD [The beneficial effect of IPL as a treatment modality for MGD has been previously reported, with significant improvement in TFLL, BUT, subjective symptom scores, and eyelid margin signs, especially for those with refractory MGD –26. The  and MGD , 29, andIn this study, we show that the IPL of Lumenis M22 with OPT, as an adjunctive therapy to MGX, is a viable therapy for BKC patients and may prevent keratoconjunctivitis recurrence by controlling blepharitis. The IPL treatment in our study patients was initiated immediately after the completion of a one-month topical steroidal treatment, and a clinical assessment that active inflammation has resolved. Though the lasting anti-inflammatory effect of the eye drops could have potentially played a role in the late improvement of eyelid signs, both patients and controls have completed the same drop regimen.Our results show that the OPT can significantly and effectively ameliorate the quality of meibum, improve MG expressibility, and regress eyelid margin signs and subjective symptoms in BKC patients more effectively than the traditional MGX therapy. As previously shown , reducinInterestingly, the OPT treatment achieved more improvement of lower eyelid signs from the second visit and thereafter, while the effect of treatment on the upper eyelids signs was more modest. One optional explanation for this discrepancy is the application of IPL at the cheek region, which lies in greater proximity to the inferior palpebral margin. A recent study by Rong et al.  reportedOur study shows that both the treatment and the control groups experienced an improvement in CFS scores, with no significant difference between the groups. This result could be related to the ongoing MGX maintenance treatment that both groups received, which is the traditional therapy for BKC, as previously reported by Rong et al. . CornealSince OPT-related uveitis and iris photoablation were previously described , 37, we Our study is limited by its small sample size, its open-label nature, a relatively short follow-up, time and strict exclusion criteria of patients with comorbidities. Our preliminary results, however, indicate that IPL with OPT therapy may have an adjunctive effect to the conventional MGX in improving the function of Meibomian glands, controlling ocular surface inflammation, relieving ocular discomfort symptoms, increasing the stability of the tear film, preventing the recurrence of BKC, and avoiding the side effect of long-term drug use. It should therefore be considered an effective adjunct treatment for BKC, specifically in the presence inflammatory skin disorders.


Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidies using cell-free DNA (cfDNA) has been widely adopted in clinical practice due to its improved accuracy. A number of NIPT tests have been developed and validated. The purpose of this study is to evaluate the performance of the Veracity NIPT test for sex chromosome aneuploidy (SCA) detection in singleton pregnancies, autosomal aneuploidy detection in twin pregnancies and evaluation of Veracity clinical performance under routine NIPT conditions in a diverse cohort.n = 305); blinded retrospective and prospective study in twin pregnancies (n = 306) and prospective evaluation of clinical performance in singleton and twin pregnancies (n = 10564).Blinded retrospective study in singleton pregnancies , 4 , 2  and 1  cases. Validation study results for autosomal aneuploidy detection in twin pregnancies exhibited 100% sensitivity and specificity and correctly classified 3 trisomy 21, 1 trisomy 18 and 1 trisomy 13 samples. Clinical performance evaluation of Veracity was performed in 10564 pregnancies with median gestational age of 13 weeks, median maternal age 35 years and median gestational weight of 64 kg. Based on confirmation feedback the PPV for trisomies 21, 18 and 13 was estimated at 100% , 100%  and 71% , respectively. Estimated PPV for Monosomy X was 57% , while the NPV for SCA detection was estimated at 100% .Veracity NIPT test is based on a very powerful, highly accurate methodology that can be safely applied in the clinical setting.The online version of this article (10.1186/s13039-019-0446-0) contains supplementary material, which is available to authorized users. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidies using cell-free DNA (cfDNA) has been widely adopted in clinical practice due to its improved accuracy, compared to traditional screening approaches using measurements of nuchal translucency and biochemical analytes. Consequently, international bodies including the American Congress of Obstetricians and Gynaecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) endorse NIPT as a routine screening option , 2.A number of NIPT tests based on targeted and whole genome-based technologies, mainly employing Next-Generation Sequencing (NGS), have been developed and validated –6. TheseMore recently, NIPT has expanded to include the detection of sex chromosome abnormalities (SCAs), and has the potential to offer significant added value to parents and physicians in the form of early detection, outcome preparation and timely treatment. NIPT is also of clinical importance in multiple gestations which pose considerably more difficult management issues, especially in relation to the risks of invasive procedures. Thus, recent studies have focused on the implementation of cfDNA analysis in this pregnancy group. The dizygotic twin group poses additional challenges, because each fetus contributes different amounts of cfDNA in the maternal circulation , 8. Thern = 305), the detection of trisomies 13, 18, 21 in twin pregnancies (n = 306) and the evaluation of clinical performance for trisomies 13, 18, 21 and X,Y under routine testing conditions in a diverse test cohort (n = 10564). Collectively, this report conveys a complete clinical picture of the Veracity NIPT test performance and assesses the implementation and clinical impact on a broader scale.This study summarizes the Veracity test validation results for the detection of sex chromosome aneuploidies (SCAs) in singleton pregnancies (n = 300). In total we detected, 286 normal, 7 Turner , 4 Klinefelter , 2 Triple X  and 1 47,XYY cases. All cases underwent confirmation by amniocentesis. In the male conceptuses, the test correctly detected 4 /4 47,XXY cases and 1/1 47,XYY case, exhibiting 100% sensitivity and specificity. In the female conceptuses the test correctly detected 7/7 45,X cases and 2/2 47,XXX cases, exhibiting 100% sensitivity and specificity (Table For the detection of sex chromosome aneuploidies (SCAs), the clinical performance of Veracity was assessed in a blinded retrospective validation study of a total of 305 plasma samples. The final analysis included samples that fulfilled the fetal fraction threshold of 4%  designed to detect fetal trisomies 13, 18 and 21, in a cohort of 306 pregnancies of at least 10 weeks of gestation. Six samples exhibited an insufficient fetal fraction. Trisomy 21 was detected in 3/3 cases, trisomy 18 in 1/1 case and trisomy 13 was detected in 1/1 case  , vanished twin and oocyte donation and demonstrated high sensitivity and specificity. Collectively, data from this work is consistent with previous validation results  and showThe targeted assay described here is inherently characterized by high depth of sequencing, which allows highly accurate fetal fraction quantification and aneuploidy detection. In the clinical setting accurate fetal fraction quantification is of paramount importance for accurate classification , 11. AccIn contrast to many previous studies, the cohort of samples in the prospective study of 10564 samples was diverse and included twin pregnancies as well as IVF and egg donor pregnancies. The study included pregnant women of all risk levels with 46.8% under the age of 35. This age distribution indicates the broader applicability of NIPT in the general population.p value < 0.00001). This finding is in accordance with previously published data [Approximately 3.7% of samples in the clinical performance study presented a fetal fraction below 4%. We observed that a high proportion of such samples was obtained from participants with higher body weight. The median weight of samples with fetal fraction below the threshold was 78 kg whereas for samples with fetal fraction above threshold was 64 kg  sensitivity for all common trisomies. Overall, demonstrated sensitivities indicate a more robust performance of Veracity compared to other methodologies, as published in multiple meta-analysis studies , 16 . ThIn the present prospective study, we determined the PPV for trisomy 13 and monosomy X to be 71 and 57% respectively. The lower PPV for trisomy 13 and monosomy X is expected and is concordant with results from previously reported studies from large scale cytogenetic analysis , 19. TheThe application of NIPT has revolutionized prenatal screening of common aneuploidies and other conditions of clinical relevance. This study provides further proof of the high accuracy of NIPT compared to conventional screening methods. The role of first trimester nuchal translucency measurement and conventional biochemical testing needs to be reassessed in the context of the use of cfDNA testing which is a powerful tool in prenatal care. Furthermore, we hereby show that Veracity, a new NIPT test based on a novel technology which was developed to overcome many of the limitations of other NIPT tests, exhibits high accuracy both in validation studies and routine testing conditions. The test’s high read depth and ability to efficiently capture cell- free DNA fragments delivers state-of the-art performance in fetal aneuploidy detection, fetal fraction estimation and cost effectiveness.With the advent and broad implementation of these powerful NIPT technologies, challenges related to pretest counseling issues, objective assessment of performance, the interpretation of results and counseling-related issues do arise and need to be addressed. Conclusively, NIPT is a powerful tool that provides clinicians and prospective parents with important information and empowers them to make informed decisions regarding pregnancy management.Blinded retrospective and prospective studies.All samples collected retrospectively were obtained anonymously from pregnant women of at least 18 years of age from the 10th week of gestation from multiple referring centres. Protocols used for sample collection were approved by the National Bioethics Committees and informed consent was obtained from all participants. All samples collected prospectively were collected after the 9th week of gestation and sent to our laboratories. Appropriate informed consent was obtained from all subjects included in this study.A total of 305 singleton pregnancy samples were included in the blinded retrospective validation study for SCAs detection. All samples were confirmed by invasive testing. Five samples exhibited insufficient fetal fraction and were excluded from analysis. The sex chromosome abnormality cases enrolled in this study included 45,X, 47,XXY, 47,XXX, and 47,XYY cases.A total of 306 twin pregnancy samples were included in the blinded validation study for aneuploidy detection of trisomy 13, 18, 21. This cohort included abnormal cases for these trisomies. A subset of 100 samples was collected in a blinded retrospective manner. All samples were confirmed by invasive testing. A subset of 206 twin samples was collected prospectively. In this cohort, confirmation feedback was available for abnormal samples. Six samples out of the 306 exhibited insufficient fetal fraction and were excluded from analysis.g for 10 min, followed by 16000 g for 10 min. Isolated plasma samples were stored at − 80 °C until subsequent analysis. Samples were coded to all operators and the analysts who processed the samples. Circulating cell-free DNA (cfDNA) was extracted from 4 ml of plasma using the Qiasymphony DSP Virus or Qiasymphony Circulating DNA Kit . Samples were processed for library preparation and were enriched using a target capture protocol as previously described using custom target capture sequences (TACS) of approximately 250 bp designed to capture selected loci on chromosome 21, chromosome 18 and chromosome 13, chromosome X and chromosome Y [A mean of 8 ml of peripheral blood was collected from each subject into EDTA-containing tubes or cell-free DNA BCT tubes . A mean of 4 ml of plasma was isolated using a double centrifugation protocol by centrifugation at 1600 Median and interquartile range (IQR) was used as descriptive statistics. The IQR is considered as a measure of statistical dispersion, being equal to the difference between the third (upper) quartile and the first (lower) quartile, with the term quartile being used to describe the division of our data set into four equal portions (quartiles). The IQR value provides useful information on the spread of those data points that (i) lie around the median and (ii) account for the 50% of the total number of data points. The larger the IQR value the larger is the spread.Custom target capture sequences (TACS) were designed to capture selected loci on chromosomes 21, 18, 13, X and Y with approximately 500 TACS per chromosome. Classification of fetal aneuploidy was performed as previously described with modifications [Results were compiled electronically and were reviewed by a subject matter expert who assigned the final classification.n = 390). Consultation was provided by our laboratory personnel for the collection of a subsequent sample. All results were reviewed by the laboratory director prior to the final reporting of results to the referring physician. Confirmation feedback and follow-up outcome was either requested by a laboratory director or provided voluntarily by the referring centres including details for the confirmation method. The confidence intervals were computed using the Clopper and Pearson method and reflect the additional uncertainty from exclusion of no-follow up cases. The assumption is that the expected value of the true proportions  do not change between the total number of positive calls and the subset for which we have follow-up.All data included in this prospective study were generated in our CLIA certified and CAP accredited laboratory until February 2018. Testing was performed in whole blood samples that were collected in cell-free DNA BCT tubes  after the 9th week of gestation and sent to our laboratories by 176 different referring centres from 21 countries. Appropriate informed consent was obtained from all subjects included in this study. Plasma was isolated as described above. Upon reception and sample processing, samples were evaluated for initial laboratory quality metrics. Samples were rejected if the tubes were broken, blood volume was too low, insufficiently labelled or if the sample was clotted, grossly hemolysed or arrived in the laboratory more than 8 days after collection. A redraw was requested for samples that failed to meet initial laboratory quality control criteria. Samples that failed quality control criteria were re-drawn and analysed. A re-draw sample was also requested for samples that failed to meet a sufficient fetal fraction threshold Additional file 2:Figure S2. Distribution of fetal fraction estimates. (TIFF 60 kb)Additional file 3:Table S1. Patient Demographics. Table S2. Test Performance. (DOCX 14 kb)


Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited.Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA.A large cross-sectional serological survey was conducted in captive elephants , sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%).This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.The online version of this article (10.1186/s12985-019-1142-8) contains supplementary material, which is available to authorized users. A number of infectious diseases significantly impact elephant population sustainability, particularly in captivity . Of greaIn Thailand, an earlier study sampled pharyngeal lymph nodes and found no evidence of EEHV in 31 Asian elephants based on PCR . More reDetermination of EEHV antibody titers to establish seroprevalence rates among various elephant populations is based on detection of antibodies against an EEHV-1A viral envelope protein, glycoprotein B (gB). Screening of Asian elephants in U.S. and European zoos showed that nearly 80% of PCR positive animals were seropositive for this protein . The aimn = 994) in private, tourist, and logging camps that were included in a health screening program under the Mobile Elephant Clinic Project in Thailand, led by trained veterinarians and staff of the National Elephant Institute (NEI), Lampang, Thailand. Animals tested represented ~ 25% of the total population of captive elephants in the country. Only elephants deemed healthy (asymptomatic) by veterinarians during the routine examinations were included in this study.The serological survey was conducted retrospectively on serum samples collected between January 2010–February 2015, and comprised elephants  at the laboratory research unit of the NEI until analysis. One blood sample per elephant was used for this cross-sectional study, and randomly selected when multiple samples were available. Serum samples were thawed, diluted 1:100 and 1:200 in phosphate-buffered saline, and assessed for the presence of antibodies using the EEHV gB specific capture ELISA described by . ResultsP-value. All potential risk factors were included in multivariable logistic regression analysis to create the model, with the exception of management type due to a high correlation with region. Region was chosen to be included in the full model because the model fit was better than the full model with management type. The AIC was used in a backward procedure to select the best model . All analyses were performed using R version 3.3.0; 2016-5-3 [A single serum dilution (1:100 and 1:200) was considered positive when the OD ratio was ≥ 3, undetectable at < 2, and inconclusive between 2 and 3 . An anim2016-5-3 .Animals were housed at 96 camps in 20 provinces throughout Thailand Fig. ; howeverStudy population demographics are presented in Table Serology results for both serum dilutions are shown in Additional file p = 0.06), but there were no differences between the age groups. Elephants from camps utilizing extensive management systems had higher seroprevalence than those managed more intensively, with some regional differences. Compared to the North, elephants in the Central, Northeast and East regions had a lower odds for a positive sample. No association was found for the EEHV status of the sample with camp cluster size and sampling period.Descriptive serology test results related to potential risk factors Table  were useIn the multivariable logistic regression analysis, only region remained in the model after backward elimination of the variables and therefore the results are the same as from the univariable model with region Table .The present study was the first to conduct a large cross-sectional survey of EEHV seroprevalence among captive elephants in Thailand. Using an EEHV-1A gB protein antigen ELISA , over 40n = 11) were found in the North. Overall, two thirds of EEHV antibody seropositive elephants were found in North, South and West regions of Thailand. By contrast, only two cases (2/32) occurred in the Central region, an area with only a few facilities close together, with limited exchange of animals from the outside. Understanding spatial differences in seroprevalence is complicated, however, by uncontrolled/unregistered elephant movements and transfers, particularly among facilities within those regions, and so needs further study.One of the characteristics of herpes viruses is their ability to go into latency. By certain unknown stimuli these latent viruses may be reactivated . If reacThe type of elephant management system was a significant risk factor to positive EEHV antibody seroprevalence in the univariable model, with 47% antibody seroprevalence in extensive systems compared to 37% in more intensive systems. The North and West regions include areas along the border with Myanmar, and contain more than half of the captive elephant population in Thailand. Although there is clinical evidence of EEHV-HD in captive elephants in Myanmar, there has been no molecular confirmation to date . However, given the close contact and/or transport of captive elephants between the Thai-Myanmar borders, transmission of the viral disease to elephants in the North and West of Thailand from Myanmar is possible, similar to what has been documented for foot and mouth disease viral transmission across these regions , 30. CapCaptive elephants in the South also had a relatively high antibody seroprevalence. In general, these were working elephants from the North and West that are taken to rest at their owner’s home in the South during the low tourist season. Conversely, elephants in the Central, East and Northeast regions live in more urban areas, closer to humans, where land and especially forest, is limited, and generally they are not exchanged between camps. The camps in intensive management systems also are less likely to transport elephants or recruit elephants from outside those regions than those in extensive systems. That may limit the degree of exposure to the virus, and agrees with our finding that most elephants living in isolated areas were seronegative. It is likely the seropositive elephants that experienced recent infection or reactivation might be related to camps with frequent or with rare viral reactivations.n = 4016 elephants), upwards of 1600 may have been exposed to the virus. The EEHV antibody seroprevalence survey showed that “region” was a significant risk factor associated with the disease incidence, particularly in the North, which is likely to be related to management or perhaps genetic relationships. Since the first diagnosed case of EEHV HD in 1999, this disease has resulted in elephant deaths, particularly calves, around the world, although it is more sporadic than epidemic in captive populations. Long et al. [Elephants sampled in this study were involved in tourism or logging, which requires tame elephants; hence, the higher numbers of adults than other age categories, and females being more prevalent than males. Trending towards significance (OR = 1.29) was a sex effect, with more males being seropositive, although the relevance of this is unknown. By contrast, elephant age, camp cluster size, and sample collection period were not significant risk factors for EEHV antibody seroprevalence. Our finding that 42.3% of captive elephants in Thailand were seropositive for EEHV antibodies suggest a high rate of viral exposure in this population. Extrapolating to the total captive population in the country , especially if they have been suspected of prior active infection.Additional file 1:Figure S1. Stacked bar plots with numbers of negative and positive animals per herd (ranked on herd size). Using as definitions for a positive sample: both OD ratio ≥ 3 (graph a) and OD ratio ≥ 4 (graph b). (PDF 58 kb)Additional file 2:Table S1. Seroprevalence based on an EEHV1A glycoprotein B protein antigen specific ELISA of elephants sampled throughout Thailand (n = 994) between 2010 and 2015, and the percentage of samples testing positive1, inconclusive2 or negative3 relative to potential EEHV risk factors based on different interpretation modalities: 1:100 dilution; 1:200 dilution. (PDF 58 kb)Additional file 3:Table S2. Univariable regression analysis of potential risk factors for the presence of EEHV antibodies in elephants sampled throughout Thailand between 2010 and 2015 (n = 994) based on an EEHV1A glycoprotein B protein antigen specific ELISA. Seroprevalence is based on strict cutoff: positive if both OD ratio’s > 4. (PDF 52 kb)


In addition, studies reporting on in vivo changes in space-flown mice were published. Finally, this Collection also brings together articles reporting experiments using cells cultured under conditions of real microgravity on the International Space Station, or exposed in ground-based facilities, in order to study morphological and molecular alterations in different cell types.A return to the Moon, Mars expeditions, and a rise in space tourism will lead to an increasing number of human spaceflights. The  However, while a space journey is fascinating, unfortunately, it can cause various short- and long-term health concerns. Human and animal bodies undergo dramatic changes after an extended stay on the International Space Station (ISS)5. It includes administering medicine to astronauts, cosmonauts, taikonauts and private space travellers in outer space and on Earth, along with the development of countermeasures for the various health problems of humans in space.Space medicine deals with the physiological and biological effects of spaceflight on the human organism, its organs, tissues and cellsg) in space. It is necessary to detect the cellular changes responsible for the various medical problems often observed in humans on the ISS. The cellular response to microgravity (µg) has been partially described for humans and animal models, whereas the intracellular regulatory mechanisms are still a mystery. Studies on whole organisms, cells and tissues are not only complementary but essential for a holistic interpretation. In such models, several molecular mechanisms involved in biological processes have been characterised6.For medical and biotechnological reasons, it is important to study animals and mammalian cells that are exposed to real microgravity  bioreactor, the random positioning machine (RPM), the magnetic levitator, and the two- (2D) and three-dimensional (3D) clinostats, to prepare for spaceflights and to perform ground-based µg experiments on stem and other specialised cells10. In addition, the National Aeronautics and Space Administration (NASA), the European Space Agency (ESA) and other agencies offer the possibility of short-term µg experiences, such as parabolic flight (PF)13 and sounding rocket missions12.Studies of tissues and cells exposed to r-µGravitational biology and space medicine’ includes publications reporting on health concerns for astronauts and cosmonauts16 during space missions, and physiological changes caused by short-term µg17. Several studies investigating the impact of µg on the behaviour and health of mice on the ISS were published in this Collection23, in addition to investigations at the cellular level in space24 and in short-term µg provided by PF maneuvers25. Moreover, cell studies in simulated (s-) µg at ground-based facilities are included in this Collection30.The Guest Edited Collection ‘et al.14 investigated nine astronauts, and showed that the composition of the astronauts’ microbiome  was changed during space travel. Notably, the altered skin microbiome might be involved in the frequently observed skin rashes and hypersensitivity episodes of astronauts. These findings warrant further detailed examination before deeper space exploration starts. Moore et al.15 demonstrated that long-duration spaceflight adversely affects post-landing operator proficiency. Eight crewmembers experienced general discomfort in motor function and motion perception after a mission, and a lack of cognitive reserve. This only manifested in duplicate tasks that recovered to baseline four days after landing15. These results suggest that the development of countermeasures should target the cumulative effect of the subtle physiological changes observed on landing day. Recommendations for countermeasures were published in Moore et al.15. Reynolds et al.16, investigated 301 astronauts and 117 cosmonauts, demonstrating that space radiation has no strong impact on their mortality. They showed that survival estimates from Kaplan-Meier curves were largely congruent with those of competing risk methods, suggesting that if ionising radiation impacts the risk of death due to cancer and cardiovascular disease, the effect is not dramatic16. However, the authors concluded that future deep space exploration might provide larger doses of space radiation, leading to a different risk profile for future astronauts and cosmonauts; therefore, it is important to continue their surveillance for potential harmful effects of such exposure16.Humans in space can experience various health problems. Immunosuppression in spaceflight increases the risk of opportunistic infections. Crewmembers are exposed to different stressors, potentially altering the composition of their microbiomes. Voorhies g-conditions revealed the similarity of postural recovery responses in 1g-, hypo- and hyper-gravity conditions17. These results showed that muscle synergies and segmental strategies acquired on Earth are robust and persistent across variable acute changes in gravitation17.Investigations of the human stance during PF maneuvers compared to 1g, and for investigating changes in different tissues23. The detailed behavioural analysis of these mice provides a useful model system for increasing our knowledge of human responses to spaceflight18. These studies provide the opportunity to examine how physical movement influences responses to µg18.The NASA Rodent Habitat on the ISS was used to study the behaviour of mice in r-µet al.19 studied the vas deferens and testes tissues of space-flown mice. They found that during the 3-week spaceflight an adaptive protein profile formed in the germinative tissue of male mice19. In addition, gene expression of Tet2 demethylase was increased and histone deacetylase (Hdac1) decreased19. The mRNA expression changes might be important during the early period of readaptation, since they could lead to an increase in the expression of target genes19.Ogneva 1. Cadena et al.20 investigated E3 ubiquitin ligase MuRF1 (Muscle-specific RING finger 1) null KO mice in space on the ISS and by the hindlimb suspension model. The muscle of MuRF1 null mice was protected in the ground-based model. By contrast, skeletal muscle atrophy in these mice was enhanced by unique mechanisms in space20.Spaceflight is associated with loss of muscle mass and strengthet al. investigated mice on the ISS21. They demonstrated blood-retinal barrier (BRB) disruption and ocular adaptation after the 35-day mission. Post-flight proteomics studies revealed changes in cell death (apoptosis), cell cycle, immune responses, mitochondrial function and metabolic stress in space-flown mice compared to ground-based control animals21. The results of this study show a complex cellular response influencing the structure of the retina and BRB integrity after the 35-day space mission21. A second study assessing eye changes after spaceflight identified 600 differentially expressed genes by RNA sequencing in mouse retinas22. The differentially expressed genes were associated with retinitis pigmentosa and changes in chromatin structure. In addition, the thicknesses of the retina, the retinal pigment epithelium and the choroid layer were lower after spaceflight, indicating that retinal changes cause the visual problems22. Finally, whole-transcript cDNA sequencing showed that a 35-day stay in orbit on ISS may alter the homeostatic gene expression of mice spleens by a combination of µg and other environmental stressors23.Another important health concern for crewmembers is spaceflight-induced structural and functional damage of the eyes. Mao g episodes provided by PFs. Gambacurta et al.24 showed that rapamycin induces transcriptional activation of human blood-derived stem cells (BDSCs) towards osteogenic differentiation in space. These findings, applying proteomics and epigenetic methods, may represent the first step in identifying new biomarkers and therapeutic targets to treat osteoporosis in space and on Earth24.The Collection also covers cell and molecular biological studies in space, and for short-term µ25. The flow and rolling speed of cells were moderately accelerated during µg periods, which were accompanied by a clear reduction in rolling rate. After the PF, whole blood tests indicated monocytes were in a “primed” state, with potentiated antigen-induced pro-inflammatory cytokine responses. In addition, anti-inflammatory cytokines were elevated, and monocytes showed a surface molecule pattern indicating immunosuppression. The results suggest an immunologic counterbalance to avoid disproportionate immune responses.A PF study examined the attachment of peripheral blood mononuclear cells to adhesion molecules under flow conditions, and antigen-induced immune activation in whole blood26. A reduced response of MSCs to priming in µg might also indicate less involvement of these cells in tissue remodelling26. Arun et al.27 studied the effects of s-µg on increasing stemness in human colorectal cancer cells (HCT116), using a rotary cell culture system (RCCS). This study highlighted the importance of µg and physical factors in the regulation of stemness in colorectal cancer cells27. Cancer research in space is currently a popular area of study29, with the prospect of researchers developing new cancer treatment strategies. Notably, Costantini et al.28 showed that RCCS-exposure promoted cells to form 3D spheroids. It also stimulated pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells28. These results are important for stem cell biology, and confirm earlier results where other cell types were exposed to µg7. Cho et al.30 examined endometrial stroma cells exposed to 3D clinorotation and showed that µg inhibited decidualisation by reducing forkhead box O3 (FOXO3a) expression, autophagic flux and proliferation, as well as decreasing protein kinase B (Akt) activity, matrix metallopeptidase 2 (MMP2) expression and migration. They suggested that µg during spaceflight might lead to an unreceptive endometrium by suppressing decidualisation potential30.3D clinorotation was used to study mesenchymal stromal cell (MSC) response to inflammatory stimulation56Fe irradiation in mice, showing specific short-term changes in neuropathology and behaviour31, with differences in dose, sex, and disease predilection. Therefore, future studies assessing the risks of space radiation exposure seem to be important for space travel moving forward.Finally, an animal study investigated the early effects of Gravitational biology and space medicine’ Collection gives a broad overview of the different space research topics (Fig. in vivo and in vitro studies investigating health problems for crewmembers, but also includes basic scientific cell experiments and studies with Euglena gracilis exposed to PF maneuvers, where hierarchical clustering showed that changes were induced by the different accelerations32.In conclusion, the ‘ics Fig. . It comp