Dichaete is the only Sox gene known to be involved in segmentation. To determine if similar mechanisms are used in other arthropods, we investigated the role of Sox genes during segmentation in the spider Parasteatoda tepidariorum. While Dichaete does not appear to be involved in spider segmentation, we found that the closely related Sox21b-1 gene acts as a gap gene during formation of anterior segments and is also part of the segmentation clock for development of the segment addition zone and sequential addition of opisthosomal segments. Thus, we have found that two different mechanisms of segmentation in a non-mandibulate arthropod are regulated by a SoxB gene. Our work provides new insights into the function of an important and conserved gene family, and the evolution of the regulation of segmentation in arthropods.Sox genes encode a set of highly conserved transcription factors that regulate many developmental processes. In insects, the SoxB gene  Insects, spiders, centipedes and lobsters all belong to a group of animals known as arthropods. A common feature of these animals is that their bodies are made up of repeated segments. However different arthropods build their segmented bodies in different ways. For example, the fruit fly makes all of its segments at the same time, while most other arthropods – including spiders – make a few segments at once and then add the rest, one or two at a time, to the rear end of their bodies.Dichaete, which belongs to the Sox gene family. However it was not known if Sox genes also control the production of segments in other arthropods like spiders.Recent research in different insects has shown that these two processes – adding segments simultaneously or sequentially – are more similar than previously thought. This research also showed that these processes involve a gene called Sox21b-1 to make both the segments that carry their legs , and the segments that make up the rear of their bodies (which are added one at a time).Paese et al. have now found that, just like insects, the common house spider does indeed require a Sox gene to form its segments. Specifically, the experiments revealed that spiders need a Sox gene called Since spiders and insects both use a Sox gene to control the formation of their body segments, it is likely that the ancestor of arthropods used one too. However, because spiders and insects use a different Sox gene for these processes, Paese et al. suggest that one gene may have replaced the role of the other during the evolution of insects and spiders.Together these findings broaden the current understanding of how genes interact to organise cells to build organisms and how these processes evolve over time. Furthermore, since Sox genes direct many important events in all animals, including humans, the discovery of a new role for one of these genes may help scientists to better understand the development of other animals too. Arthropods are the most speciose and widespread of the animal phyla, and it is thought that their diversification and success are at least in part explained by their segmented body plan . In termDrosophila melanogaster and the short germ beetle Tribolium castaneum. However, studies of other arthropods including the myriapods Strigamia maritima and Glomeris marginata, and chelicerates, such as the spiders Cupiennius salei and Parasteatoda tepidariorum, have provided important mechanistic and evolutionary insights into arthropod segmentation (hedgehog (hh) and orthodenticle (otd) expression (hunchback (hb) and distal-less (dll) , which in turn is required for oscillatory expression of pair-rule gene orthologues including even-skipped (eve), and runt (run)  . In contnt (run) . Interesnt (run) . TherefoDichaete is required for correct embryonic segmentation in the long germ insect D. melanogaster, where it regulates pair-rule gene expression  family gene pression . Interesastaneum . This siastaneum . Howevere latter .P. tepidariorum , another Group B Sox gene  in the pedipalps and L1, and Deformed-A (Dfd-A) in L1  in L1 . HoweverA) in L1 . In the A) in L1 : all legA) in L1 . In ClasA) in L1  and we tA) in L1 , but subA) in L1 . Since mA) in L1 .Sox21b-1 depletion on cell death and proliferation at stages 5 and 9 between knockdown and control embryos using antibodies against Caspase-3 and phosphorylated Histone 3 (PHH3) (Sox21b-1 knockdown embryos (n = 10) (Sox21b-1 knockdown embryos (We next examined the effect of 3 (PHH3) . At the (n = 10) . At stag(n = 10) , but we  embryos . However embryos .Sox21b-1 knockdown embryos show decreased cell proliferation compared to controls (n = 10 for each)  . Interesferation . Thus, oP. tepidariorum, decapentaplegic (dpp) and Ets4 are required for cumulus formation   . To inveSox21b-1  is involectoderm   gene is involved in mesoderm specification  and, via cad, the expression of pair-rule gene orthologues including eve  and hh. At stage 9 en is expressed segmentally from the cheliceral to the O3 segment in control embryos  underlying the formation of the SAZ and the sequential addition of segments from this tissue. We show that Sox21b-1 acts upstream of Wnt8 and Delta-Notch signalling in this GRN and is necessary for the activation of these important signalling pathways during posterior development . Cocoons from mated females were removed and a small number of embryos were immersed in halocarbon oil for staging .To identify the phylogenetic relationship of ClustalW   equipped with an Axiocam 506-Mono and a colour digital camera were used. Immunostained embryos were imaged with Zeiss LSM 800 or 880 with Airyscan confocal microscopes. For live imaging, embryos were aligned on heptane glue coated coverslips and submersed in a thin layer of halocarbon oil. Bright-field live imaging was performed using an AxioZoom V16 stereomicroscope, while fluorescence live imaging was performed with confocal microscopes. Image stacks were processed in Fiji  and HeliFragment of genes were amplified using PCR and cloned into pCR4-TOPO . Oligonucleotide sequences are listed in Immunostaining was carried out as described previously  with minP. tepidariorum Sox21b-1 were isolated from the 1134 bp coding sequence of the gene: fragment 1 spanning 549 bp and fragment 2 covering 550 bp. Double stranded RNA for P. tepidariorum Dl (853 bp), Wnt8 (714 bp) and the coding sequence of GFP (720 bp) as used previously (Double stranded RNA (dsRNA) for parental RNA interference was synthesized according to , dissolveviously , were tr In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Patricia Wittkopp as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Nikola-Michael Prpic (Reviewer #1); Natascha Turetzek (Reviewer #2).Thank you for submitting your article "A SoxB gene acts as an anterior gap gene and regulates posterior segment addition in a spider" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Sox21b-1, during the embryonic development of the spider Parasteatoda tepidariorum. In insects where the role of SoxB family genes in segmentation was examined, it was reported that a particular member of this gene family, called Dichaete, was involved. The present paper shows that although Dichaete is present and conserved in spiders, it is not involved in segmentation; instead, another SoxB paralog called Sox21b-1 is involved in spider segmentation. The authors show that the gene is maternally provided and is then expressed in an anterior and a posterior domain in the forming embryo. They have also tested the function of the gene by RNAi and find that the RNAi knockdown animals show a striking phenotype where the entire posterior portion of the germ band is missing. Interestingly, a milder phenotype shows a "gap-gene-like" phenotype. The authors therefore go on to investigate the influence of Sox21b-1 RNAi on known gap-like factors and posterior dynamic factors. They convincingly show that Sox21b-1 is required for the expression of the known gap-like factors but is also required for the expression of the posterior dynamic patterning system that operates in the segment addition zone. Thus, with this work the authors provide the first factor that integrates the central gap system with the posterior dynamic system of spider segmentation. In fact, Paese et al. have identified a gene that is required for the development of the entire opisthosoma, and acts upstream of the previously identified system of caudal, Delta-Notch, and Wnt8 signalling. Moreover, the authors provide data for a mechanism in which Sox21b-1 orchestrates posterior segment addition by organising the germ layers and the specification of mesendodermal cells. Finally, this study provides insight about how certain gene regulatory interactions can change during evolution following gene/genome duplication events.The authors have studied the role of a SoxB related gene, Essential revisions:Sox21b-1 RNAi more carefully. The expression might be reduced/lost because the input of Sox21b-1 is missing. However, the expression might also be lost simply because the cells/tissue/organ where the gene is usually expressed are reduced/lost . By using whole-mount in situ hybridisation as the only indicator of target gene expression, it may be difficult in some cases to distinguish why the expression is reduced/lost in the RNAi animals and this may lead to erroneous statements about gene regulatory interactions between the studied genes. Remarkably, all of the studied (likely) target genes presumably respond "negatively" to Sox21b-1 RNAi (i.e. their expression is reduced or lost), which increases the likelihood that not all of these "negative responses" are due to true genetic interactions. To clarify this, a marker could be used that co-expresses with one  of these presumed targets, but that is not regulated by Sox21b-1 to show that the cells are there and their identity properly specified. This would strengthen the authors’ claim that Sox21b-1 is upstream and solidify their model in Figure 6. Alternatively, clonal RNAi in embryos could be performed, that would provide "control" cells directly next to injected RNAi cells. As these experiments would, however, require extensive additional work, the authors are therefore asked to carefully rephrase those portions of the text that deal with this issue of gene regulation and expressly state that the reduction/loss of gene expression after RNAi may have other causes than gene interaction and therefore not all of the genes shown in the manuscript to respond negatively upon Sox21b-1 RNAi may be true regulatory targets.1) The authors should interpret the missing expression of some likely target genes after 2) The authors provide a very detailed staging of all the presented embryos, however the decision exactly which developmental stage is present is not always clear from the pictures especially in severely affected RNAi embryos. Please provide the criteria for staging of the RNAi embryos in the Materials and methods section. Was the staging based on certain landmarks or developmental processes of the normal developmental table? If yes, how was the decision made when these landmarks were lost after gene knockdown? Or were the RNAi embryos staged by developmental time? Essential revisions:1) The authors should interpret the missing expression of some likely target genes after Sox21b-1 RNAi more carefully. The expression might be reduced/lost because the input of Sox21b-1 is missing. However, the expression might also be lost simply because the cells/tissue/organ where the gene is usually expressed are reduced/lost . By using whole-mount in situ hybridisation as the only indicator of target gene expression, it may be difficult in some cases to distinguish why the expression is reduced/lost in the RNAi animals and this may lead to erroneous statements about gene regulatory interactions between the studied genes. Remarkably, all of the studied (likely) target genes presumably respond "negatively" to Sox21b-1 RNAi (i.e. their expression is reduced or lost), which increases the likelihood that not all of these "negative responses" are due to true genetic interactions. To clarify this, a marker could be used that co-expresses with one  of these presumed targets, but that is not regulated by Sox21b-1 to show that the cells are there and their identity properly specified. This would strengthen the authors´ claim that Sox21b-1 is upstream and solidify their model in Figure 6. Alternatively, clonal RNAi in embryos could be performed, that would provide "control" cells directly next to injected RNAi cells. As these experiments would, however, require extensive additional work, the authors are therefore asked to carefully rephrase those portions of the text that deal with this issue of gene regulation and expressly state that the reduction/loss of gene expression after RNAi may have other causes than gene interaction and therefore not all of the genes shown in the manuscript to respond negatively upon Sox21b-1 RNAi may be true regulatory targets.Sox21b-1 knockdown may be difficult to interpret and, as the reviewers recognise, would involve a significant amount of new experimental effort that, in the end, may not be conclusive. However, as mentioned above, we do accept the reviewers point that effects we see may not be direct and, therefore, as requested, we have rephrased the relevant text to clarify that not all of the genes that respond negatively to the Sox21b-1 RNAi may be regulatory targets and that indeed the observed effects could be indirect through Sox21b-1 regulation of other intermediate genes or through the incorrect specification of germ disc cells. For example, in the Discussion section, we have added: “Note that while it is possible that Sox21b-1 could regulate Delta and Wnt8 and other segmentation genes directly or via intermediate factors, it remains possible that the loss of expression of these genes upon Sox21b-1 RNAi could be an indirect consequence of the loss of cells or incorrect cell specification when Sox21b-1 expression is knocked-down.”The reviewers raise an important point. While our data shows that Sox21b-1 acts upstream of the segmentation genes we have tested, we did not mean to suggest that these genes are directly regulated by this transcription factor. Indeed, this regulation could well be several steps upstream of these genes and Sox21b-1 regulation could even be indirect by specifying the cells that will express these genes. Unfortunately, we do not know of a suitable marker that is expressed in the specific germ disc cells at the relevant stages that would be able to help with this point as suggested by the reviewers. With regard to embryonic RNAi injections, we believe that clonal analysis of We have also tried to make this clearer in Figure 6 and the legend of this figure.2) The authors provide a very detailed staging of all the presented embryos, however the decision exactly which developmental stage is present is not always clear from the pictures especially in severely affected RNAi embryos. Please provide the criteria for staging of the RNAi embryos in the Materials and methods section. Was the staging based on certain landmarks or developmental processes of the normal developmental table? If yes, how was the decision made when these landmarks were lost after gene knockdown? Or were the RNAi embryos staged by developmental time?This is a very good point raised by the reviewers. Therefore, we have added a more detailed explanation of the criteria used for the fixation and staging of the RNAi embryos to the Materials and methods section. In summary, even though the embryos were immersed in oil for visualization, it was sometimes difficult to stage embryos due to their severe phenotypes . Consequently, we relied on developmental staging by hours, and the morphology of sibling embryos from the same cocoon that did not show the phenotype (i.e. from cocoons where the effect was not fully penetrant) and at least in the case of older embryos, by the appearance of the limbs.


Background: A major concern for clinicians in prescribing medications to pregnant women and neonates is the possibility that drugs might have damaging effects, particularly on long-term brain development. Current understanding of drug permeability at placental and blood-brain barriers during development is poor. In adults, ABC transporters limit many drugs from entering the brain; however, little is known about their function during development.Methods: The transfer of clinically relevant doses of paracetamol (acetaminophen), digoxin and cimetidine into the brain and cerebrospinal fluid (CSF) was estimated using radiolabelled drugs in Sprague Dawley rats at three developmental stages: E19, P4 and adult. Drugs were applied intraperitoneally either acutely or following chronic exposure (for five days). Entry into brain, CSF and transfer across the placenta was measured and compared to three markers  that cross barriers by “passive diffusion”. The expression of ABC transporters in the brain, choroid plexus and placenta was estimated using RT-qPCR.Results: All three drugs entered the developing brain and CSF in higher amounts than the adult brain and CSF. Comparisons with “passive” permeability markers suggested that this might be due to age-related differences in the functional capacity of ABC-efflux mechanisms. In adult animals, chronic treatment reduced digoxin  and paracetamol  entry compared to acute treatment, with the decrease in digoxin entry correlating with up-regulation of efflux transporterabcb1a (PGP). In fetal and newborn animals, no gene up-regulation or transfer decreases were observed. Instead, chronic paracetamol treatment resulted in increased transfer into the fetal brain .Conclusions: These results suggest that the developing brain may be more at risk from acute drug exposure than the adult brain due to reduced efflux capacity and at greater risk from chronic treatment due to a lack of efflux mechanism regulatory capacity. ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; CSF, cerebrospinal fluid; DPM, disintegrations per minute; E, embryonic ; i.p., intraperitoneal; i.v., intravenous; MRP, Multidrug resistance-associated protein; P, postnatal; PGP, P-glycoprotein; RT-qPCR, Real time quantitative polymerase chain reaction; SD, standard deviation; µCi, micro Curie.et al., 2008; Saunderset al., 2018). For lipid soluble compounds, transcellular transfer of many compounds is limited by efflux transporter mechanisms . This information is essential for understanding the likelihood of a drug to enter the brain directly from the fetal circulation (once the substance has crossed the placenta) or from the circulation of a newborn , who lacks placental protection. In the clinic, drugs are administered to pregnant women and newborns for a range of conditions and over 1200 drugs have been prescribed during pregnancy and lactation  and will be further considered in the Discussion. To determine the relative contributions of the placental and brain barriers in drug protection of the developing brain, individual measurements in fetal blood and cerebrospinal fluid (CSF) are required. Access of any molecule into the CNS is determined by: (i) their physicochemical properties, such as molecular size and lipid solubility; (ii) biological properties ; and (iii) physiological properties of brain barriers that are developmentally regulated (e.g. CSF secretion). The actual level that a molecule reaches in brain and CSF is also influenced by the turnover of CSF  but also on their specificity, if any, for individual ABC transporters that play an active role in molecular exclusion at the barrier. It is this last function that is little understood during brain development but is critical to understand their possible limitation of drug entry into the brain at different stages of its maturation.Animal studies on the entry of drugs into the developing brain following administration to pregnant animals or newborns are also very scarce. The few studies that have been published on pregnant rodents have been reviewed in SaundersA key biological problem, which has implications for understanding potential deleterious effects of drugs administered to pregnant women or new-born infants, is the stage of brain development when the efflux transporters appear and when they become functionally effective. It is not necessarily the case that once a transporter is present it will show the same level of activity throughout development; this might increase or decrease at different times (see Discussion andabcb1) to digoxin transfer  and BCRP (abcg2) to cimetidine transfer (abcc3) may limit paracetamol entry via its glucuronidated metabolite (abcg2) or other MRP (abcc) transporters. All three drugs are available in a radiolabelled form. The use of radiolabelled drugs is essential for detection of a drug in the very small volumes of plasma and CSF available from fetal and neonatal rodents.The present paper describes experiments using three index drugs that are given to pregnant women and/or neonates . Two of the drugs selected (digoxin and cimetidine) are used for their peripheral therapeutic effects  but there is some evidence that they do enter the brain to a limited extent in adult animals digoxin:. These tThe results showed that there are clear age-dependent differences in the entry of the three drugs  into brain and CSF. In addition, over the course of chronic exposure, drug entry was reduced only after a certain stage of brain maturation. Some of these differences appear to be accounted for by changes in the expression levels of individual ABC transporters. The study provides a basis for future comprehensive research into the entry of a wide range of drugs into the developing brain.All procedures involving animals were approved by the University of Melbourne Animal Ethics Committee  and conducted in compliance with Australian National Health and Medical Research Guidelines. All efforts were made to ameliorate any suffering of animals. They were handled by experienced researchers in such a way as to minimise stress prior to being anaesthetised. All animals were assessed as healthy prior to commencement of experiments. Animals were monitored prior to and following every injection ensuring there was no abnormalities in weight (>15%), appearance  or behaviour .Animals. Sprague Dawley rats were supplied by the University of Melbourne Biological Research Facility and housed in groups of 2–4 (adult) or full litters per cage , on a 12 h light/dark cycle withad libitum access to food  and water. Age groups investigated (at treatment completion) were time mated pregnant females at E19 (350-400g body weight), postnatal pups at P4 and non-pregnant female adults (175-230g body weight). E19 was chosen because this is a stage of development when adequate volumes of blood and CSF can be obtained for analysis from fetal rats without pooling . In chronic experiments, doses of unlabelled drug were given twice daily (09.00 and 17.00h) for four days before a final injection on the 5th day identical to acute experiments. In passive marker experiments, all dosing was acute and contained only the [3H]- or [14C]-labelled compound .In all experiments involving postnatal animals, injections were intraperitoneal (i.p.). In pregnant animals for fetal drug entry studies, the final injection of the radiolabelled marker was given intravenously (i.v.), as the peritoneal cavity required opening prior to the sampling period to gain access to the fetuses. For passive markers , fetal animals were individually injected i.p. while still within the uterine horn. All experiments took place between 09.00 and 15.00h. All pregnancy studies were completed with a single treatment group on one day. Postnatal groups were conducted with a single treatment group on one day or with the chronic group completed prior to the acute group.Sample collection. Samples were collected 30 minutes after the final injection. This duration was chosen partly to limit any potential metabolism of the drugs and markers used but also to allow enough time for i.p. injected markers to reach the blood stream and, from there, to access the brain parenchyma across brain barriers, as well as to limit the CSF sink effect that could influence drug levels  and cortical segments of brain tissue dorsal to the ventricle of the frontal/parietal lobes, as previously described (For adult and newborn (P4) experiments, animals were terminally anaesthetized using inhaled isofluorane . Blood samples were collected from the right cardiac ventricle, CSF from the cisterna magna . Animals were placed on a 33°C heating plate in a supine position and an endotracheal catheter inserted to maintain a clear airway. The femoral artery and vein of the left hindlimb were cannulated to provide access for collecting arterial blood samples and intravenous (i.v.) injection of the radio-labelled drug or marker solution; the cannula was flushed with 1ml of heparinized  saline. All injections were made by slow infusion. Starting at 30 minutes after injection blood, CSF and brain samples  were collected serially from each fetus. At the time of each fetus sampling, the state of the placental circulation to the fetus was assessed from the colour of the umbilical blood vessels (pink veins indicate reasonable oxygenation). Time matched blood samples (200μl) were collected from the maternal arterial cannula to establish levels of radioactivity in the maternal circulation at the time of each fetal sampling. Maternal blood volume was maintained by intraarterial injection of equivalent volumes of heparinized sodium chloride solution, which also served to maintain the patency of the cannula. Fetuses were sampled over a period of 30 minutes - 2.5-hours post-injection, the time during which placental circulation to the fetuses remained adequate.Sample preparation. Samples were processed immediately after collection. Blood was centrifuged at 7000rpm for five minutes and the plasma separated. CSF samples were examined microscopically for traces of red blood cells and discarded if contaminated . In all experiments, the radioactivity in the injectate was also measured to confirm the uniformity of the injected material. All samples were weighed and transferred into scintillation vials. Soluene350  was added to the brain samples. After being incubated at 36°C overnight to allow the tissue to solubilize, two drops of glacial acetic acid (Sigma) were added to neutralize the strongly alkaline Soluene350. All samples were then mixed with 5ml of scintillation fluid  before being transferred into the liquid scintillation counter  to count radioactivity disintegrations per minute (DPM) over five minutes each with luminescence correction on. Blank vials containing the same tissues without radioactivity were also counted alongside the samples to establish the level of background counts, which were always subtracted from the radioactivity counts of the corresponding experimental samples.Sample analysis. Background-corrected DPM data from the liquid scintillation counter were normalized to the weight of the samples and expressed as DPM per µl or µg of sample. Ratios denoting brain and CSF transfer for all animals , except that no radioactive tracers were included. Before tissue collection, all surgical instruments were cleaned with RNaseZAP (Thermo Fisher Scientific) to destroy any RNases. For the E19 group, the tissue collected from each pup was brain (see Drug Entry section) and placenta. The small lateral ventricular choroid plexuses were also collected and pooled from all pups into one sample. Tissue collected from P4 and adults (brain and choroid plexus) were processed individually. The samples were placed into sterile cryogenic vials, snap frozen in liquid nitrogen and then transferred into a -80°C freezer for storage.RNA extraction and reverse transcription. RNA was extracted using RNeasy Plus Mini Kits  for the brain samples in the postnatal animals and E19 placenta samples according to manufacturer’s specifications. For the fetal brain samples and all choroid plexuses, RNA was extracted using RNeasy Micro Kits  according to manufacturer’s specifications. RNA purity and quantity were assessed using a Nano-drop  and the concentration of all samples standardized in nuclease-free water (<300ng/μl). Each RNA sample was then converted to cDNA using the High Capacity RNA-cDNA kits  containing 9µl of RNA in Nuclease-free water, 10µl 2x reverse transcriptase buffer mix (RT buffer) and 1µl 20x RT enzyme mix, making a total volume of 20µL. RNA was converted to cDNA using a thermocycler  set at 35°C for 60 minutes, followed by 95°C for five minutes and then incubated at 4°C until collection. The more stable cDNA was stored at -80°C until ready to analyse with the RT-qPCR system .RT-qPCR. Regulation of gene expression was measured using RT-qPCR and amplification detected with SYBR Green fluorescence. Primers listed inabcc1, which was at 400nM to avoid reads in no-template control (NTC) wells. PCR efficiencies were validated. The final 10µl reaction volume contained 2µl 1/10 diluted cDNA and 8µL SYBR Green master mix . No-template controls (NTCs) were prepared and analysed alongside the cDNA triplicates with every run. NTCs produced no signal, or the occasional negligible signal (> 38Ct). Plates were evaluated using the Applied Biosystems Quantstudio6 Flex machine: two minutes at 50°C, 10 minutes at 95°C, 40 amplification cycles for 30 seconds at 95°C and one minute at 60°C. Transcript counts were compared to a housekeeper peptidylprolyl isomerase B (ppib) using the equation 2-ΔCt.Group permeability data (brain/plasma and CSF/plasma concentration ratios) for the acute and chronic conditions at all ages are presented as mean ± standard deviation (SD). Microsoft Excel 2011 was used for statistical analysis. Statistical differences between the acute and chronic groups for each drug were determined by unpaired, two-tailed Student’s t-test with p<0.05 accepted as significant and F-tests to confirm equal variance.in vivo drug studies were compared with gene expression data to establish which ABC transporters changed their expression following chronic exposure, in order to see if they correlated with observed changes in drug entry results. The transporters studied were:abcc1-5 (MRP1-5),abcg2 (BCRP) andabcb1a/abcb1b . For quality control, the radioactivity counts estimated in different compartments in all experiments are shown inabcc1 (MRP1), whereas for protein-based experimentation, the protein name will be listed with associated gene in brackets, e.g. MRP1 (abcc1). The full raw data are available asUnderlying data  or multiple doses (chronic experiments) of one of three drugs  and analysed at E19, P4 and adult (see Methods). Results fromDigoxin. In acute digoxin experiments  experiments  expression . In contrast, at E19, no significant up-regulation was observed in brain cortex. Instead, chronic digoxin treatment resulted in a significant down-regulation ofabcb1b (PGP) expression . At P4, the response to chronic drug exposure in the brain was more variable. For all three drugs there was a down-regulation ofabcc2  following chronic paracetamol  andabcc5 (MRP5) following chronic digoxin  and chronic cimetidine  exposure. In the choroid plexus samples at all three ages, regulation in response to chronic drug exposure appeared to be variable. Regulation was different within each age for the three drugs, as well as for each drug between ages . The brain/plasma . The brain/plasma and CSF/plasma ratios of the hydrophilic markers sucrose and L-glucose were very low at all ages, whereas the ratio for the more lipophilic glycerol was about 50% at E19 (46.8% brain and CSF) and approached 100% at P4 (82.2% brain and 95% CSF) and in adults (85.5% brain and 103.9% CSF). In separate experimentation, 60-minute glycerol concentrations ratios reached approximately 80% in the brain and nearly 100% in the CSF at E19 (see Discussion), making values very similar to those of P4 and adult. The comparison of the brain/plasma . Therefore, a comparison was made between the drug permeability results in this study with measurements of “passive” markers of similar molecular size . These markers are thought to not bind to any influx or ABC efflux transporters and have varying lipid solubility . In contrast to E19, the chronic treatment regime in the adult resulted in reduced entry of this drug  andabcg2 (BCRP) were up-regulated . In contrast,abcc1 (MRP1) was down-regulated following chronic treatment with digoxin or cimetidine  andabcb1b (PGP) was down-regulated only following digoxin treatment .Despite no changes being observed in transfer of paracetamol, digoxin or cimetidine across the placenta following chronic treatment, some differences were observed in ABC-transporter expression . In placIn the E19 animals, passive markers (L-glucose and glycerol) indicated the extent to which placenta was able to restrict passive entry of small water-soluble markers from maternal blood and into the fetal circulation  during tInet al., 1982;et al., 2008; Saidijamet al., 2018) in response to chronic drug treatment.In this study we aimed to determine the level of entry into the brain and CSF of three drugs at different ages. We also assessed the age-dependent functional capacity of brain barriers to prevent or limit the entry of potentially harmful drugs and how this may alter with a longer treatment regime. There is only limited information available about the expression and cellular distribution of key barrier defences such as ABC transporters in the developing brain, either in the human embryo/fetus . Comparison of drug entry and passive marker entry allows interpretation to take account of possible effects of CSF turnover.Permeability of the drugs into the brain and CSF was compared to that of similarly small-sized molecules, but with different diffusion coefficients and that are not actively transported, i.e. they enter the brain and CSF by “passive” diffusion only. This provides a comparison that is useful in evaluating the level of restriction of the drug’s penetration at different ages . As indicated above, the level of drug or marker attained in the brain in these experiments is a measure of “apparent permeability” rather than absolute permeability because of the influence of the turnover of CSF, which is much less in the developing brain . However, neither fetal blood nor CSF were sampled and the brain level of the drug  was related to maternal blood or to other fetal tissues. This makes it difficult to assess the contribution of the different barrier interfaces  that may be contributing to limiting entry of the drugs into the developing brain. The present study provides information on this, as both the maternal and fetal blood and CSF were sampled in addition to the brain tissue.There are very few published studies of drug permeability of the developing brain when they were administered to pregnant rats or mice. Of these, fluoxetine and venlafaxine administered via drinking water between E0 and E10 could not be detected in the brains of the embryos , which results in greater efflux capacity of compounds (abcb1). Increased brain/plasma ratios for digoxin have been reported in wild type mice when co-administered with a PGP inhibitor and also when administered tomdr1a/1b (PGP) knockout mice, indicating that this transporter is of particular importance in limiting entry of digoxin into the brain  suggested that levels of radiolabelled digoxin (compared to the rest of the fetus) were higher earlier in gestation when fetal levels ofabcb1a (PGP) were lower. Smitet al. (1999) reported a fetal brain to maternal plasma ratio of 25% (4hrs) and 100% (24hrs) after i.v. injection of radiolabelled digoxin in pregnant mice. These would have been overestimates of the brain level, as the maternal blood level would fall progressively following injection. This type of experiment requires a dosage regime that maintains an approximately constant blood level of marker  substrate. As this up-regulation  only occurred in adults and not at P4 or E19, the results are consistent with those described byabcb1a expression in the adult brain but not earlier in development, suggesting that for a range of molecular inducers, regulation of blood-brain barrier defences may be age-dependent.Digoxin has generally been found to be a substrate for PGP (abcg2) as its efflux mechanism. Experiments in dually perfused rat placenta suggested BCRP (abcg2), but not PGP (abcb1a/b), involvement in cimetidine efflux (abcg2 (BCRP) expression in the rat brain have also been linked to differential transfer of cimetidine (abcg2 (BCRP) mRNA expression was significantly less at E21 compared to E18, so presumably some other protective mechanisms may have been involved. In the present study, in acute experiments, cimetidine entry into the adult brain was similar to that of digoxin (13%) but entered the CSF to a higher ratio (12% compared to digoxin 4%). The higher degree of cimetidine transfer into the CSF compared to digoxin could be due to less ABC transporter efflux capacity at the choroid plexus or from its lower lipid solubility, allowing greater partition into the aqueous CSF fluid. There do not appear to be any cimetidine-induced changes in efflux capacity at different ages, as no differences in transfer were detected in chronically compared to acutely treated animals. Consistent with this finding are the RT-qPCR results, showing only limited changes in efflux transporter expression following chronic cimetidine treatment  and the family of MRPs  up-regulated at the P4 brain andabcb1b (PGP) up-regulated in the adult choroid plexus  glutathionation, making it likely to interact with BCRP , the turnover of CSF (sink effect) and the effectiveness of a specific transport mechanism if present; the latter could be inward, as in the case of amino acids and a small number of drugs, but generally outward for drugs that are substrates for ABC efflux transporters.The level of a drug or other marker in brain following administration depends on the duration of the experiment, its diffusion coefficient (D), its lipid solubility  appears not to be a general property of the barrier for all molecules as glycerol did not follow this trend. This, combined with the distance of the three lipid soluble drugs below the line of passive transfer , indicatabcb1a, abcb1b, abcg2 andabcc1-5 in varying quantities, as has been shown in several published studies . There were only a few small changes in expression in these transporters following chronic treatment with the drugs (abcc1 (MRP1) andabcg2 (BCRP), while digoxin and cimetidine caused a down-regulation ofabcc1. However, these changes were not reflected in changes in placental transfer. This result may be of clinical importance as it demonstrated differences in the regulation of ABC transporters expression that are tissue specific: up-regulation with associated decreases in functional transfer into the brain, but not in the placenta.In our experiments we have been able to obtain an estimate of the placental contribution to the overall protection of the fetus from drugs administered to the pregnant mother by comparing directly the drug levels in fetal and maternal plasma. The fetal/maternal plasma ratios varied between ~30% for cimetidine treatment to ~40% for paracetamol and digoxin ;Table 8e drugs . Chronicth day was 21% for the mother and 45% for the fetus. Thus, in chronic treatment, paracetamol may have become less effective for the mother, but potentially more harmful for the fetus as more paracetamol reached its brain. If the same situation applies in patients, it would suggest that, where possible, duration of treatment should be as short as possible.Taking into account the level of drug exclusion provided by the placenta for all three drugs, the transfer from maternal blood to fetal brain in acute experiments was at a relatively similar concentration ratio as that from maternal blood to the maternal brain. Digoxin transfer into maternal brain was 12%, whereas the transfer from maternal blood to fetal brain was 17%. Similar results were seen for paracetamol  and cimetidine . In contrast to the acute experiments, different patterns of transfer were observed into the maternal and fetal brains following chronic treatment. Cimetidine, which did not up-regulate ABC transporter expression or decrease its transfer with chronic treatment, had the same ratio of transfer into both the maternal and fetal brains. Digoxin, however, decreased its entry into the maternal brain during chronic treatment, while the entry across the placenta and into the fetal brain remained the same. Paracetamol showed decreased entry into the maternal brain, while the amount entering fetal brain increased. For acute paracetamol treatment, transfer into brain  was 30% for mother and 28% for the fetus, but after five days of twice-daily paracetamol, the transfer of a dose on the 5This study has been restricted to two treatment conditions; namely, a single acute dose and twice daily chronic treatment over a period of five days. The latter corresponds to about one third of gestation. It is possible that with an even longer period of treatment, greater effects on ABC transporter expression correlated with greater restriction of drug entry in fetuses, neonates and adults would have been observed. We studied only one dose concentration of each drug but this was chosen to be in the clinical range. It is possible that with smaller doses, a lower rate of entry would be obtained. This could be the case particularly in our paracetamol experiments at E19; it seems likely that the greater entry of paracetamol may have been due to the capacity of the efflux transporters for this drug having been exceeded.et al., 2011) that has been used for a lot of developmental studies.We have estimated drug entry using only radiolabelled drugs, rather than direct measurement of unlabelled drugs, as the amounts of fetal/postnatal fluids would have been too small for other methods such as high-performance liquid chromatography (HPLC). However, the similarity of our results in adult rats for paracetamol and those ofOur results show that at the doses used, all three drugs entered the brain at all three ages studied. The entry of all of the drugs was highest in the youngest animals (E19). This is probably a combination of the negligible turnover of CSF at this age , allowing greater accumulation and possibly lesser activity of the relevant active efflux transporters. The entry into brain was also appreciably higher for paracetamol than the other two drugs at all three ages. The finding of entry of digoxin and cimetidine into the brain, particularly at E19, suggests that these drugs should be studied for possible long-term effects on brain development and behaviour of offspring. However, a much greater entry of paracetamol suggests that experiments to test for such effects in the offspring would be particularly appropriate, since this is the drug most commonly taken by pregnant women .Data are available under the terms of the The rationale for this study is the potential for transfer of medicines into the fetal and infant brain, a subject that is little understood through lack of research. It is a relevant question as there is considerable exposure of the undeveloped brain to medicines via administration to pregnant women, breast feeding women and directly to infants. The focus is upon the ABC transporter family, efficient at protecting the adult brain from lipid soluble medicines, but about which little is known in the developing brain. This question is addressed by using labelled digoxin, cimetidine and paracetamol, substrates for 3 different ABC transporters. Transport into brain and CSF is measured using radioactively labelled drug, dosed acutely or chronically and at different stages of brain development. Expression patterns of the transporters are examined at different stages of animal development and correlations sought with the transport of drugs into brain/CSF. There were large contrasts between E19 animals and P4/Adults for brain/plasma and CSF/plasma ratios for all three drugs,  with E19 having greater transport/permeability. Useful information regarding ratios of marker compounds with a range of octanol-water coefficients, drug ratios and transporter expression for acute and chronic treatment was obtained. The ‘system’ showed potential for investigation of new medicines, prioritising studies of drug action on brain development on the basis of uptake into brain, e.g. paracetamol’s greater propensity for transport into brain at E19, unravelling protective effects of transporters and adaptions to componentry of the ‘system’ with dosage, duration of therapy and drug combinations. The work is well done, clearly described, well illustrated and fairly presented.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Summary-Study Objectives:           This is a very interesting and unique study performed by an excellent group of researchers with high expertise in the field. Overall, the study provides novel data describing the permeability of three commonly used drugs in the clinic, digoxin, cimetidine and paracetamol, into the blood, brain and CSF compartments of Sprague Dawley rats at three developmental stages. These data are further complemented by qPCR-based RNA expression studies for relevant ABC transporters in the placenta, brain and choroid plexus. The findings highlight a link between novel age-specific differences in transporter regulation and penetration of these drugs into blood, brain and CSF compartments. The work significantly advances the knowledge in the field as there is a very important lack of information related to this topic.Overall Comments: The supporting background information described in the introduction and discussion is a very thorough representation of existing literature, and gives context to the results. Overall, the studies are carefully planned and the methods applied are robust. Limitations of the study are thoroughly described in the discussion. This work sets the stage for further investigation into the topic, and future directions are highlighted by the authors. The work significantly advances the knowledge in the field as there is a very important lack of information related to this topic.Specific Comments:Additional details on the drug dosing would be helpful especially since there is ample literature describing the use of the drugs in animal models. It is clear that the dosing was adapted from the Australian Medicines Handbook; however, the authors should clarify if allometric scaling or existing literature was used to adapt the drug doses for rodent studies. The authors should also address whether the observed drug plasma concentrations in their animal model reflect clinical relevant plasma concentrations.The authors should consider the extent of drug plasma protein binding when they address the various factors regulating drug permeability across the various tissue compartments; only free drug will be able to permeate across biological membranes by diffusion.Drug concentration in biological compartments is the result of the interplay of passive diffusion across biological membranes, influx/efflux transport, and drug metabolism. While the authors have nicely characterized the role of efflux transporters, they have not fully addressed the potential role of influx transporters in the permeability of the drugs studied. Members of the Solute Carrier (SLC) superfamily could be involved in the uptake of the drugs across the various compartments. The expression of these transporters could also be very variable at the different development stages. This could be further addressed in the discussion for completeness.     Measurement of radiolabeled drug in the tissue compartments will reflect total drug/metabolite concentrations rather than parent drug; hence drug metabolites may present a confounding factor in the interpretation of the data.  This should be addressed by the authors.uronidation” “glutathionylation”.In the discussion section, p17 second paragraph, please correct the spelling of the terms “gluc  I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. As I scanned this manuscript (ms), I thought the introduction rather long. On reading it, however, I was impressed how well it laid out the field succinctly, identified some of what was missing, how this report would eliminate one deficiency, and then what was found. Well done, indeed. The design is more than appropriate with the technically-sound assessment of multiple drugs and multiple passive permeability markers The methods and analysis are presented in sufficient detail for others to attempt replication. I was at first frustrated that I could not find the number of animals identified in the text. Then I found Tables 1 and 2, which, on consideration, seemed eminently reasonable given the numbers that were appropriately listed for each age group for each assessment. The authors used Tables 3 and 4 to identify materials used. Yes, the authors provided all the information needed. The 4 data tables  and the 7 figures present all the data needed very well. The authors clearly document changes to support their conclusion that the functional capacities of efflux transporters at the barriers are developmentally regulated. I did NOT think that any revisions are called for.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Additionally, we provide estimates of the proportion of the population living in communities above certain sanitation coverage levels (objective 2).The impact on diarrhoea of sanitation interventions has been heterogeneous. We hypothesize that this is due to the level of prevailing faecal environmental contamination and propose a Faecal Contamination Index (FAECI) of selected WASH indicators -level were analysed according to the identified thresholds, at country-, regional- and overall level.Objective 1: We found a non-linear association between estimated environmental faecal contamination and sanitation interventions’ impact on diarrhoeal disease. Diarrhoea reductions were highest at lower faecal contamination levels, and no diarrhoea reduction was found when contamination increased above a certain level. Objective 2: Around 45% of the population lives in communities with more than 75% of coverage with basic sanitation and 24% of the population lives in communities above 95% coverage, respectively.High prevailing faecal contamination might explain interventions' poor effectiveness in reducing diarrhoea. The here proposed Faecal Contamination Index is a first attempt to estimate the level of faecal contamination in communities. Much of the world's population currently lives in faecally contaminated environments as indicated by low community sanitation coverage. •An index for estimating faecal contamination in the community is proposed that combines a broad set of WASH indicators.•Faecal community contamination is an important determinant of a sanitation intervention's effectiveness to reduce diarrhoea.•Above a certain level of faecal contamination, interventions are less likely to lead to reductions of diarrhoea.•Less than half of the global population lives in communities in which coverage with basic sanitation services exceeds 75%. These iRecent large, well-designed and well-conducted WASH trials and intervention studies, which included sanitation improvements with high intervention fidelity, did however not yield expected effects on diarrhoeal and nutritional outcomes , which mIt has long been argued that the - measurable - impact of WASH interventions on diarrhoea depends on the number and type of faecal pathogens that people are exposed to through a variety of transmission pathways in their homes and communities  Fig. 1)Fig. 1). There is consistent and considerable evidence that entire communities benefit from sanitation improvements in individual households, e.g., . The heaThis paper is motivated by two main objectives: The first is to estimate faecal environmental contamination post-intervention from sanitation intervention reports and to use these estimates to explain heterogeneous impacts on diarrhoeal disease. For this purpose we propose for the first time a Faecal Contamination Index (FAECI) of selected WASH indicators. The second objective is to provide country, regional and overall estimates of the proportion of the population living in communities above a certain level of coverage with basic sanitation services with a focus on low- and middle-income countries to complete data on faecal environmental contamination in general and on WASH indicators used for the FAECI.22.12.1.11.practice open defecation or unsafe disposal of child faeces (S1),2.use or have access to basic sanitation services, i.e., improved sanitation facilities that are not shared between two or more households (S2),3.use or have access to safely managed sanitation services, i.e., basic sanitation services that ensure safe disposal or safe transport and treatment of excreta (S3),4.use or have access to basic drinking water services, i.e., water from improved sources that require no more than 30 min to collect water from (W1),5.use or have access to safely managed water services, i.e., improved sources accessible on premises, which provide water free from contamination and available when needed (W2),6.have access to basic handwashing facilities, i.e., handwashing facilities with soap and water on premises (H1),7.wash hands with soap after potential faecal contact (H2).A set of WASH indicators was selected to develop a Faecal Contamination Index (FAECI), on the basis of biological plausibility, demonstrated association with diarrhoeal disease , data avAn eighth indicator (S4) refers to community sanitation coverage, i.e. the proportion of the population within a community that uses or has access to basic sanitation services. The eight indicators are shown in Indicators S2, S3, S4, W1 and W2 refer to either use of or access to services dependent on the information available from the study report. S1 and H2 are indicators of behaviours whereas H1 assesses access to services. More information on the indicators and scoring of interventions is provided in The first six indicators are assessed regularly (S1: only open defecation) through nationally-representative household surveys and are compiled and reported globally by the JMP every two years . Child f2.1.2The potential level of faecal environmental contamination using the FAECI was estimated in sanitation interventions identified in a recent systematic review . This re2.1.3Relative risk estimates for diarrhoeal disease prevalence or incidence were extracted from a recent systematic review  or direc2.22.2.1We searched PubMed (in January 2018) combining both MeSH terms and keywords in order to identify studies assessing health effects from improving sanitation services at various community coverage levels . We restSanitation coverage in a community is defined here as the proportion of people in a community that use basic sanitation services. A community has been defined as a “group of people with diverse characteristics who are linked by social ties, share common perspectives, and engage in joint action in geographical locations or settings” . Communi2.2.2We searched the JMP household survey data repository in July 2017 for relevant microdata at PSU-level from Demographic Health Surveys (DHS)  or Multi2.2.3Data were analysed taking account of cluster sampling in survey data collection (using the svy-command in Stata). To calculate the percentage of the population living in communities above a defined threshold of coverage with basic sanitation services by country, two analysis steps were performed: first, the proportion of the population within the PSU using basic sanitation services was calculated. Second, the mean proportion of the national population living in clusters having at least a certain basic sanitation coverage level was calculated weighted by the mean of population weights by PSU.Confidence intervals at country-level were generated using the standard error of the mean at country-level . RegionaWe are following guidelines for accurate and transparent health estimates reporting  and have33.13.1.1A total of 17 intervention studies were included . Of thosThe frequency of reporting of the eight WASH indicators post-intervention in the intervention group in the 17 sanitation studies is shown in 3.1.2Using meta-regression analysis, estimated faecal environmental contamination as represented by the score of the FAECI and the squared FAECI score were strongly associated with the relative risks of diarrhoea of intervention studies (p = 0.006). The model including all studies explained 53% of the between-study variance. Additional binary indicators of urban versus rural setting and sanitation versus combined WASH interventions, and a continuous indicator for the difference of the FAECI between intervention and control group (the “delta FAECI”) were not significantly associated with diarrhoea risk and did not change the association of the other variables in the model.Predicted relative risks from the meta-regression model ranged from a minimum of 0.32  at a FAECI of three  to 0.94  at a FAECI of 11 . The majority of interventions with a FAECI score between 8 and 13 did not show a significant effect of diarrhoea and hence also the predicted relative risks from the model were equivalent to no effect . At a FAECI of 14–16 predicted relative risks declined again due to two studies that show low relative risks at a high FAECI , a.Fig. Using studentized deleted residuals, one study  was idenThe delta FAECI, included separately and in combination with its squared term, was not significantly associated with the effect sizes of the interventions.3.23.2.1From an initial list of 3800 citations, we selected 145 studies for abstract review and subsequently 49 studies for full text review. Of those, five studies reported the association between community coverage thresholds and diarrhoeal disease impacts . Based oFurthermore, there was evidence for an association between community coverage with basic sanitation and malnutrition , infecti3.2.2From the JMP data repository we identified a total of 111 countries for which survey microdata on use of basic sanitation services at PSU-level were available. The countries and territories included cover 29 out of 31 low-income, 47 out of 52 lower middle-income, 33 out of 57 upper middle-income and 2 (Trinidad and Tobago and Uruguay) out of 55 high-income countries and territories .Table 5NSurveys had been conducted between the years 1998 and 2017 (mean and median at 2012). 78% (87/111) of surveys were collected after 2010. The included countries cover 78% of the world population and 92% of the population in low- and middle-income countries. A complete list of countries included in the analysis, the survey type, year, region and income status are provided in 3.2.3The percentage of the population living in communities in which more than 75% and 95% of the population are covered with basic sanitation services by country is given in 44.14.1.1The analysis shows a non-linear association between the estimated level of faecal contamination in the community, as assessed by the FAECI, and impacts of sanitation interventions on diarrhoeal disease. It suggests that sanitation interventions are more effective at reducing diarrhoea at lower levels of faecal contamination, and that interventions are less likely to show diarrhoea reductions if faecal contamination in the community remains above a certain level.4.1.2The percentage of the population from low- and middle-income countries living in communities with more than 75% and 95% coverage with basic sanitation services is estimated at 45% and 24% respectively. Large regional discrepancies exist, with less than a third of the African and South-East Asian population living in communities covered with >75% basic sanitation services.4.2Faecal environmental contamination was assessed combining a set of WASH indicators that cover many household- and community-level transmission pathways for diarrhoeal disease (compare to Some of the WASH indicators measure behaviours (S1 and H2) while another access to or presence of specific infrastructure (H1) or both depending on the study's reporting . Improving use of or access to services requires different intervention strategies. Examples include health risk communication and education for improving use  and subsThe analysis suggests that settings with low faecal contamination post-intervention are more likely to show impacts on diarrhoeal disease than settings with high prevailing faecal contamination. In poor settings, which often carry the burden of considerable faecal contamination of the environment, sanitation and WASH interventions are crucially important, and should be considered as a necessary but possibly insufficient step for achieving disease reduction . In the We had initially planned to validate the FAECI on another WASH category and attempted to extract data on the FAECI indicators from 14 drinking water filtration interventions. However, none of the filtration intervention studies reported whether sanitation facilities were safely managed, the presence of a handwashing facility with soap and water or observed handwashing with soap. While most filtration studies reported the presence of latrines or toilets it was often not clear whether these facilities were of an improved or unimproved technology. This underlines the importance of a minimum set of WASH indicators that should be reported in all WASH intervention studies independent of the WASH category addressed.The FAECI should be regarded as a first attempt to simply, transparently and reasonably estimate the level of faecal contamination in a community environment in a way that is comparable across various intervention locations. We believe that even with slightly different indicators or different scoring, the general results and broad conclusions would be similar. We had – for example - constructed a different version of the index that included a combined measure of unsafe disposal or presence of child and animal faeces, which was however not pursued due to data scarcity on animal faeces or animal presence. Analysis of the estimated faecal contamination assessed with this modified index led to basically the same results as the here proposed one  of mostly low- and middle-income countries. Furthermore, even though most surveys were conducted recently, 16 surveys data back to before 2010 .Due to data constraints at cluster-level of national household surveys, estimates of community sanitation coverage are estimates of coverage with basic sanitation services and not necessarily safely managed services. In 2015, the JMP estimated that 68% of the world population used basic sanitation services of which only 39% were estimated to be safely managed . CommuniNot all previous evidence showed that greater community sanitation coverage leads to positive health outcomes . One of 5A large proportion of the world's population lives in communities that are vulnerable to significant faecal contamination from poor management of excreta. We propose a first attempt to estimate the level of faecel contamination in an intervention setting. Results of the analysis suggest that WASH interventions are more likely to lead to reductions of diarrhoeal disease when faecal contamination of the living environment has been reduced below some threshold level. This underlines the importance of interventions that reach whole communities assuring that everybody uses a safe toilet and sanitation system that separates excreta from human contact along the whole sanitation chain. WASH interventions may show no impact on diarrhoea because of persisting faecal contamination due to for example incomplete community coverage or use of sanitation facilities that do not safely contain faecal material or treat it to a level suited to the end use or disposal type. Such interventions might nevertheless be necessary interim steps to reduce faecal environmental contamination for achieving disease reduction in the future. The assessment of a minimum set of WASH indicators is useful for the evaluation of the prevailing major pathways of faecal transmission and of the intervention's effectiveness.None.


Nanostructured surfaces and nanoparticles are already widely employed in many different fields of research, and there is an ever-growing demand for reliable, reproducible and scalable nanofabrication methods. This is especially valid for multifunctional nanomaterials with physical properties that are tailored for specific applications. Here, we report on the fabrication of two types of nanomaterials. Specifically, we present surfaces comprising a highly uniform array of elliptical pillars as well as nanoparticles with the shape of nanopockets, possessing nano-cavities. The structures are fabricated by nanoimprint lithography, physical and wet-chemical etching and sputter deposition of thin films of various materials to achieve a multifunctional nanomaterial with defined optical and magnetic properties. We show that the nanopockets can be transferred to solution, yielding a nanoparticle dispersion. All fabrication steps are carefully characterized by microscopic and optical methods. Additionally, we show optical simulation results that are in good agreement with the experimentally obtained data. Thus, this versatile method allows to fabricate nanomaterials with specific tailor-made physical properties that can be designed by modelling prior to the actual fabrication process. Finally, we discuss possible application areas of these nanomaterials, which range from biology and medicine to electronics, photovoltaics and photocatalysis. Nanostructured surfaces and nanoparticles are widely employed in many fields of research and technology, and there is an ever-growing demand for reliable and reproducible nanofabrication methods. In biology and medicine, nanostructured surfaces and nanoparticles are employed both because of their optical ,2 or magA huge variety of different nanomaterial fabrication methods can be found in literature ,15,16,17Next to nanostructured surfaces, it was also shown that nanoparticles can be fabricated by combining NIL and physical vapor deposition or spin-coating techniques ,29,30,31In the present study we show that UV-NIL can be used to fabricate an array of nanoparticles, starting from a uniform array of elliptical pillars, and that these nanoparticles can be transferred to solution yielding a nanoparticle dispersion. The so fabricated nanoparticles have the shape of nanopockets as each of them possesses a cavity. Furthermore, multifunctional optical and magnetic properties of the nanopockets were obtained by incorporating different materials. Scanning electron and scanning force microscopy, as well as optical transmittance measurements give experimental evidence of the successful nanofabrication. Finally, we also show optical simulation results which are in good agreement with the experimentally obtained data. This also means that the optical nanopocket properties can be tailored to specific demands by predictive modelling prior to the fabrication process. In conclusion, we present a highly versatile nanofabrication method that allows nanomaterials to be produced for a wide range of applications, which are discussed in more detail in the final section.2 and contains about 4.2 × 108 pillars. From the master, a hybrid stamp composed of polydimethylsiloxane (PDMS) and hard PDMS (h-PDMS) was fabricated by methods reported in literature  and treated with an anti-sticking layer . The master shows a regular pattern of elliptical pillars. The pillars have a height of 250 nm and an elliptical base with a major axis of 400 nm and a minor axis of 200 nm. The pillars are separated by a gap distance of 200 nm from each other. The so structured area extends over 1 × 1 cm2 for 1 min at a wavelength of 365 nm). The stamp was removed, and the imprinted sample placed on the hotplate for a hard bake process at 150 °C for 1.5 min. The following reactive ion etching was performed in an atmosphere of 15 at% O2 at a pressure of 0.24 mbar for 2 min to remove the residual layer of uncured resist. The wet-chemical etching process was done with a 1:1 dilution of a metal ion free developer  in ultra-pure water and an immersion time of the sample of 30 s at room temperature. The sample was then thoroughly rinsed with ultra-pure water and dried under a nitrogen stream  was spin-coated at 4000 rpm (100 nm film thickness) and hard baked at 200 °C for 5 min on a hotplate. The organic UV-curable imprint resist layer  was spin-coated at 4000 rpm (200 nm film thickness) and soft baked at 100 °C for 1 min. The imprint was carried out by pressing the h-PDMS/PDMS stamp into the imprint resist and curing with a self-built high-power UV-LED lamp  with a target to substrate distance of about 100 mm. Au and NiFe (Ni:Fe 80:20 at%) were sputtered in pure Ar atmosphere at a gas pressure of 2 µbar and at a power of 20 W and 40 W, respectively. TiOTM). The second was applied for transmittance measurements due to its transparency (article number 56002 from TIXO®). The third one was applied for a transfer of the nanopockets into solution due to its water solubility .For the transfer of the nanopocket array from an opening-down to an opening-up configuration, three different commercially available adhesive tapes were employed. One tape was applied to allow for sputter deposition and electron microscopy due to its vacuum stability . This process was repeated three times.Scanning electron microscopy (SEM) images were recorded on a Zeiss Supra 40 electron microscope at 5 kV acceleration voltage using the in-lens detector. The nanostructures on the Si substrate were imaged directly on the substrate. The images of the opening-up nanopocket array were taken after sputter deposition of a 20 nm Au layer for the generation of a conductive top layer to allow for SEM. The nanopockets from the solution were prepared for imaging by drop-casting onto a Si wafer substrate with a native oxide surface and letting the solution dry at room temperature.TM).The sample surface topography was characterized by scanning force microscopy (SFM)  in acoustic mode with PPP-NHCR tips  equipped with an additional visible light source and a Si photodetector. For polarization control of the incident light beam, a linear polarizer was inserted in the light path . The measurements were recorded at an incident light beam normal to the sample surface and referenced to the substrate material. The transmittance curves obtained were smoothed and plotted using commercially available software from OriginLab Corporation, Northampton, MA, USA.Optical simulations were conducted using a commercial software based on the finite-difference time-domain (FDTD) method . The complex refractive indices of Au and glass were taken from literature ,38. TranThe magnetic moment was measured by a Lake Shore Cryotronics Inc Westerville, OH, USA 8600 series vibrating sample magnetometer (VSM). The nanopockets were drop-casted from aqueous solution onto a silicon wafer substrate and let dry resulting in a random nanopocket orientation. Measurements were carried out with the external magnetic field oriented in-plane of the substrate at room temperature.The nanostructure fabrication process is schematically shown in 2. With an inverse imprint structure of the same master, it was recently shown that a very narrow size variation of the imprinted structures is achieved, resulting in a standard deviation of the area of the elliptical base of +/−3% [In the following, we will give experimental evidence of the nanostructures fabricated by UV-NIL and sputter deposition  are appIn conclusion, the fabrication technique of the nanostructured surfaces as well as of the multifunctional nanopockets presented here provides a powerful tool for nanofabrication due to its simplicity, flexibility and scalability. A wide range of different nanomaterials with tailor-made physical properties can be realized. Thus, many different applications may greatly benefit from the fabrication technique and its versatility.


The advent of diffusion compartment imaging (DCI) to probe tissue microstructure in vivo holds promise to map reactive microglia using MRI scanners. But this potential has never been demonstrated. In this paper, we performed longitudinal DCI in rats that underwent dorsal root axotomy triggering Wallerian degeneration of axons—a pathological process which reliably activates microglia. After the last DCI at 51 days, rats were sacrificed and histology with Iba-1 immunostaining for microglia was performed. The fraction of extra-axonal restricted diffusion from DCI was found to follow the expected temporal dynamics of reactive microgliosis. Furthermore, a strong and significant correlation between this parameter and histological measurement of microglial density was observed. These findings strongly suggest that extra-axonal restricted diffusion is an in-vivo marker of reactive microglia. They pave the way for MRI-based microglial mapping which may be important to characterize the pathogenesis of neurological and psychiatric diseases.Reactive microgliosis is an important pathological component of neuroinflammation and has been implicated in a wide range of brain diseases including brain tumors, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and schizophrenia. Mapping reactive microglia  More generally, neuroinflammation is an increasingly recognized important pathological pathway in a variety of psychiatric disorders and has become a promising target for treatment7. Reactive microgliosis also has a pivotal role in the pathogenesis of brain tumors such as glioblastomas wherein tumor cells attract and then activate microglia8.The presence of reactive microglia in neuroinflammation is thought to play a key role in the pathogenesis of a range of brain diseases such as Alzheimer’s diseasein vivo is therefore becoming increasingly important to investigate the pathogenesis of brain diseases and to assess response to treatment. To avoid the invasiveness and sparsity of biopsies, investigators have relied on positron emission tomography (PET) with specific ligands to map reactive microglia2. However, the cost and availability of PET scanning prevent its widespread use and its limited resolution precludes the identification of neuroinflammatory loci or specific inflamed brain circuits.The ability to effectively and efficiently map the presence and status of microglial cells 10—a novel approach based on diffusion-weighted magnetic resonance imaging (DW-MRI) to image the microstructure of neural tissues. Unlike diffusion tensor imaging (DTI), DCI considers that there are multiple microstructural environments in each voxel and provides a separate parameterization for the signal arising from each of them11. The ability of DCI to detect glial cells has been theoretically suggested13 but has never been demonstrated.A promising alternative to PET scans to map reactive microglia is the use of diffusion compartment imaging (DCI)14. Wallerian degeneration is the pathological process that occurs following axonal injury and includes axonal loss, myelin clearance, and the recruitment of reactive microglia. Its temporal evolution is well established14 and a wealth of studies have demonstrated the sensitivity of DTI to axonal loss and myelin clearance21. The consensus that emerges from these studies is that radial diffusivity starts increasing weeks after injury and is a marker of myelin clearance, while axial diffusivity decreases in the acute phase before normalization and is a marker of axonal loss. DTI studies however have failed to map reactive microglia.A reliable empirical setup to investigate reactive microglia mapping is to trigger Wallerian degeneration in the spinal cord of rats10, a recently developed DCI model, to longitudinally characterize the tissue microstructure of seven rats (3 control and 4 injured) imaged at baseline, and at 4, 13, 37, and 51 days after unilateral dorsal root axotomy (or a sham intervention). After 51 days, rats were sacrificed and we compared the last DIAMOND parameters to histological measurements of microglial density. The two-compartment DIAMOND model used has one compartment that captures the axonal signal (represented by parameters that have their DTI counterpart) and one compartment that captures the extra-axonal restricted diffusion thought to arise from reactive microglia.In this study, we used DIAMONDfear) of DIAMOND and histological microglial density are presented.This section first presents the histological validation of the pathological features occurring in our experimental setup. Next, the temporal and spatial evolutions of DIAMOND parameters are presented. Finally, the correlation between the fraction of extra-axonal restricted diffusion  51 days after the injury  is observed in the late phase only  and diffusivity (dear) of the compartment representing extra-axonal restricted diffusion follow parallel and opposite temporal evolutions , one with a mostly late component (detected by changes in fascicle-specific λ⊥), and one with both an acute component and an exacerbation in the late phase (detected by fear and dear).These findings suggest that there are three distinct temporal processes at play: one with a mostly acute component (detected by changes in fascicle-specific fear) is a marker of reactive microglia, we analyzed the correlation between fear extracted from DIAMOND 51 days after surgery and the microglial density extracted from histology with Iba-1 immunostaining. On the ipsilateral side, both fear and the microglial density increase closer to the lesion  whereas on the contralateral side, both parameters remain broadly constant throughout the region of interest  as used in this study has been defined specifically to capture the diffusion of water molecules trapped in closed isotropic environments such as reactive mircroglia. Unlike axons, microglial cells do not favor a particular direction of diffusion and are morphologically more isotropic23. The use of an isotropic tensor (as used in some studies25) rather than a closed sphere with fixed radius (as suggested in others12) for modeling this compartment is justified by the presence of microglial cells featuring a broad range of diameters. The cumulative signal arising from all these individual restricted compartments is therefore well represented by a Gaussian distribution, which leads to a tensor in our signal formula. This is analogous in DTI to the use of a cylindrical tensor rather than a cylinder to model the cumulative signal arising from a bundle of individual axons.The strong correlation between et al.26. In the latter, the diffusion coefficient of the isotropic compartment is set to the free diffusivity of water molecules at 37 °C and is meant to represent water freely diffusing in the extracellular space and the cerebrospinal fluid (CSF). In the absence of histological validation, the underpinning pathological processes leading to raised extracellular free water remains speculative. Neuroinflammation27 and white matter atrophy28 have been hypothesized. Of note, the characteristic displacement of water molecules for such a free water diffusion coefficient (D0 = 3 μm2/ms) with common values of the effective diffusion time (μm (=μm. Assuming axons roughly 1 μm in diameter (d), such a characteristic free displacement (x) would only occur with an axonal density of about 0.0005 (=32), the so-called extracellular free-water fraction might actually be an imperfect proxy to the extra-axonal restricted diffusion.Importantly, what is referred to as the extra-axonal restricted compartment in the present study is different from the extracellular free-water mapping developed by Pasternak dear decreases , the dynamics of its Wallerian degeneration may resemble that of other cranial nerves rather than the slower dynamics observed in the rest of the central nervous system. This would explain the discrepancy with studies on the optic nerve19. Second, the type of lesion used to elicit Wallerian degeneration may affect its timing. While rhizotomy was used in the present study, ischaemia19 and contusions18 were used in others. Third, the radial diffusivity in studies where no other parameter was available may have confounded multiple microstructural changes that are independently captured by the fraction of extra-axonal restricted diffusion (fear) in the present study. Supporting this claim is the fact that the dynamics of the radial diffusivity in two studies20 exhibits a two-stage increase similar to the dynamics of the extra-axonal restricted diffusion in the present study. Furthermore, in the study by Zhang et al.20, only the correlation between radial diffusivity and Luxol fast blue optical density at 30 days after injury is reported to be significant while no significant correlation is reported at 38 hours, 3 days, and 7 days. The authors acknowledged that the changes in radial diffusivity occurring in earlier stages might therefore be due to pathological processes other than myelin clearance. The discrepancy in the temporal evolution of the radial diffusivity therefore appears to be spurious and our findings in fascicular axial and radial diffusivities corroborate previous findings from DTI studies.The other findings from the temporal evolution of DIAMOND parameters corroborate previous findings from DTI studies. The time course of the DIAMOND axial diffusivity is remarkably similar to that of the DTI axial diffusivityfear correlates with the morphometric aspects of reactive microglia —not its other functional aspects. Reactive microgliosis is itself a complex pathological process which involves microglial phenotypic changes, and production of cytokines and chemokines. A note of caution is therefore in order: While the fraction of extra-axonal diffusion (fear) appears to be a DCI marker of reactive microglia, it is not sufficient to fully characterize reactive microgliosis. The latter depends on the microglial specific phenotype and pathological status. For instance, in glioma, the tumor environment leads to a change in microglial phenotype which is different from the inflammatory phenotype and microglia activated in that way does not release proinflammatory cytokines but present other transcriptional changes8. Tissue analysis  thus remain of paramount importance if the goal is to more comprehensively characterize reactive microgliosis. The two techniques are therefore complementary. While DCI enables whole-brain/spinal cord mapping of reactive microgliosis , tissue analyses enable detailed characterization of the reactive microgliosis in one specific area . Future developments may explore the use of fear mapping to guide biopsy by informing the investigator of potential loci of interest.The present study has two main limitations. First, the fraction of extra-axonal restricted diffusion 20) prevents the definite one-to-one association of DIAMOND parameters and pathological features during the course of Wallerian degeneration. The claims in this paper rather relied on two sources of evidence: (i) the empirical comparison of DIAMOND parameters and histology at the final time point (51 days post-injury), and (ii) the comparison of the time course of DIAMOND parameters with the known dynamics of Wallerian degeneration from the literature. Future work will investigate longitudinal histology with immunostaining and comparison with DIAMOND parameters to investigate whether the early increase in the fraction of extra-axonal restricted diffusion indeed represents early recruitment of microglial cells.Second, the absence of longitudinal histology  model, for the Female Long Evans rats  were used. Two groups were defined: a control group (3 rats) and an injured group (4 rats). Both groups were anesthetized using a rodent anesthesia system  with vaporized Isofurane  and underwent laminectomy at L2–L3 to expose the spinal cord. The injured group then underwent left unilateral rhizotomy from L2 to L3 performed with a microciser.The rhizotomy model chosen is a selective section of the dorsal and ventral rootlets with no injury to the spinal cord. Therefore the sectioned axons are clearly identified and the extension of the lesion is controlled, predictable and reproducible. In this work, we focus on the lesion extending rostrally along the ascending fibers of the gracile fasciculus. The absence of direct spinal cord injury precludes the formation of intra-spinal hematoma and cavities that can be highly variable in contusion and section models. Furthermore, hematomas and the subsequent blood remnant create long term artefacts and can be responsible for poor image quality in the vicinity of the lesion site. The spinal cord damage in total spinal transection is often too extensive to allow a precise identification of the lesioned axons and would have prevented the use of the contralateral side as a control.Following rhizotomy, muscles were sutured together and the skin was sutured. Post-operative care included subcutaneous administration of Baytril Enrofloxacin , buprenorphine , and lactate ringer solution . The protocols were approved by the ethical committee for animal care of the health science sector of the Université catholique de Louvain. All methods were performed in accordance with the relevant guidelines and regulations.DWI were acquired in injured rats before and 4, 13, 37, and 51 days after surgery. In control rats, only the pre-surgical and the 51-day post-surgical acquisitions were performed. For each image acquisition, rats were anesthetized with an isofurane-air mixture (2.5% for induction and 1–1.5% for maintenance). Rats were then placed in a dorsal decubitus position on a custom-made bed. Respiratory rate and rectal temperature were continuously monitored with a physiological monitoring unit . The body temperature was maintained constant at 37 °C with a circulating warm-water pad.b0 image) and 6 shells of 36 gradient directions each at b-values: 300, 700, 1500, 2800, 4500, 6000 s/mm2. The voxel resolution was 0.1 × 0.1 × 1 mm3. The other acquisition parameters were TE = 23 ms, δ = 4.5 ms, and Δ = 12 ms. The orientations of diffusion vectors were maximally separated within and across shells using Caruyer’s method34. The duration of each acquisition for each rat was around 2.5 hours. Correction for animal motion and Eddy current was achieved by affine registration of each scalar DWI to the b0 image. To this end, images were resampled to a resolution of 0.05 × 0.05 × 0.06 mm3 to allow improved multi-scale pyramidal registration.A TriPilot sequence coupled with respiratory gating was acquired before DWI acquisition and the experimenter confirmed (and corrected if needed) adequate positioning of the rats. All acquisitions were carried out on an 11.7 Tesla Bruker BioSpec MRI . The transmitter volume coil (diameter 72 mm) and the receptive surface coil  were coupled for the acquisition covering the T12-L4 vertebral segments. The magnet was shimmed to adjust the homogeneity using quadratic adjustment. The multi-shell DWI acquisition sequence included 18 non-weighted DW images .DIAMOND considers the presence of different microstructural environments in voxels. It represents the DWI signal at the voxel level as a weighted sum of signals arising from each of them. The weights represent the contribution of each environment to the DWI signal. In this work we focus on imaging the rat spinal cord and use a two-compartment DIAMOND model with one isotropic restricted and one anisotropic compartment. The isotropic restricted compartment captures the signal arising from water molecules that do not show a preferential direction of diffusion. It is represented by a single diffusion coefficient D and a heterogeneity parameter D is itself parameterized by a fascicular radial diffusivity λ⊥ and a fascicular axial diffusivity λ‖ which are akin to their widely-known DTI counterparts. The parameter D10. We let fear be the fraction of the DWI signal explained by the isotropic compartment so that 1 − fear is the contribution of the anisotropic compartment.The anisotropic compartment is represented by a continuous statistical distribution of diffusion tensors with a mean tensor b-value b and a gradient direction g, the signal attenuation S/S0 modeled by DIAMOND reads:For a 2 and a gradient orientation at a angle <70° from the mean axonal orientation were filtered out. The parameters fear, dear, D were then estimated at every voxel from the input DWI data using the method described in10.At high b-values and with gradient directions aligned with the mean axonal orientation, the signal attenuation is such that it falls below the noise level and brings no relevant information about the tissue microstructure. To filter out these DWI and stabilize the estimation of the DIAMOND parameters, a DTI model was first estimated to determine the mean axonal orientation of the spinal white matter. DWI acquired with both a b-value > 4500 s/mmb0 image. The hypo-intense signal of the anterior spinal vein on b0 image and the high FA of the corticospinal tract on DTI were used as landmarks to manually segment the gracile fasciculus. Vertebral levels were identified on an anatomical T1-weighted image acquired within the same acquisition. The ROI did not include any voxels close to the cerebrospinal fluid. The result is an ROI volume of approximately 200,000 voxels with in-plane segmentation. The corresponding levels on histology were determined by direct comparison with an atlas35.We defined a region of interest (ROI) covering the T12  to L2 (at the superior border of the lesion site) levels of the gracile fasciculus as follows. The cerebrospinal fluid was identified and the spinal cord was manually segmented on the μm-thick sections using a cryostat .Fifty-one days after surgery, all rats were euthanized and transcardially perfused successively with phosphate-buffered saline (PBS) and with 4% phosphate-buffered formaldehyde to fix the tissues. After the perfusion, the spinal cords were extracted and post-fixed in cold 4% buffered paraformaldehyde overnight. The following days, the tissues were successively transferred to 10%, 20%, and 30% sucrose/PBS (w/w) baths maintained at 4 °C for one night each. The spinal cord pieces were then frozen and stored at −80 °C, cryopreserved in optimum cutting temperature (OCT) compound, and sliced axially in 20 18 and counterstained with Cresyl Violet for the Nissl substance. Immunohistochemistry with the anti-panneurofilament SMI-312  for neuronal cell bodies20 and Iba-1 in combination with a secondary immunoperoxidase stain  for reactive microglia20. The staining was performed with Diaminobenzidine (DAB) substrate (Sigma-Aldrich).Sections were stained with Luxol Fast Blue (LFB) for myelin integrity assessmentet al.36 and Phoulady et al.37 was developed as follows. A range filter with a radius of 5 pixels was first applied to the greyscale images to highlight areas with sharp contrast (as are the neurites) using the function rangefilt. Multi-level thresholding was then applied to the filtered image using the multithresh function with default parameters and with four levels38. Pixels with intensity values above the maximum level were considered part of the neurites (for SMI-312) and microglia (for Iba-1) while pixels with values below this threshold were assigned to the background. The ratio of the number of neurite-labeled (respectively microglia-labelled) pixels over the number of background pixels was defined as the neurite density . In each slice of the ROI, the mean myelin intensity, neurofilament density, and microglial density were computed for the ipsilateral and contralateral side in the injured group and for both sides simultaneously in the control group.LFB, SMI-312, and Iba-1 images were transformed to greyscale intensity values using the rgb2grey function of MATLAB 2015a . The LFB intensity is referred to as the myelin intensity. To assess the fraction of the volume occupied by neurofilaments and reactive microglia, a Matlab routine borrowing ideas from Puigvert Histological findings were compared between the control group and the ipsilateral and contralateral side of the injured group. Values from the intensity of LFB staining, from neurofilament density, and from microglial density were averaged on each side and in each slice within the gracile fasciculus so that one value was recorded for each side of each histological slice of each rat. For the control groups, both sides were averaged together.p-values and rejection of the null hypothesis was based on the corrected p-values.To assess the temporal evolution of DIAMOND parameters, the values at all spinal levels rostral to the lesion within the gracile fasciculus were used. At each time point, the value between the contralateral and ipsilateral sides were statistically compared. Since comparisons were performed at 4 time points and for 5 parameters, a Bonferroni correction for multiple comparisons (with 20 comparisons) was applied to the fear from DIAMOND, the fear maps were first downsampled so that the number of DIAMOND slices equaled the number of histological slices. The values of microglial density and fear were averaged in each slice and on each side of the gracile fasciculus independently. The Pearson correlation coefficient between glial density and fear was then computed over all slices. Downsampling the fear maps was preferred over upsampling the histological values as the latter would artificially inflate the sample size leading to artificially low p-values.Finally, to compare microglial density from histology and t-tests and rejection of the null hypothesis was based on a threshold at All two-sample tests were two-sample