We sampled 20 taxa across a broad phylogenetic, body size, and physiological scope from the Atchafalaya River Basin of Louisiana as an environmental analogue to the Late Cretaceous coastal floodplains of North America. Samples were analysed for stable carbon, oxygen and nitrogen isotope compositions from bioapatite and keratin tissues to test the degree of ecological resolution that can be determined in a system with similar environmental conditions, and using similar constraints, as those in many Mesozoic assemblages. Isotopic results suggest a broad overlap in resource use among taxa and considerable terrestrial–aquatic interchange, highlighting the challenges of ecological interpretation in C3 systems, particularly when lacking observational data for comparison. We also propose a modified oxygen isotope-temperature equation that uses mean endotherm and mean ectotherm isotope data to more precisely predict temperature when compared with measured Atchafalaya River water data. These results provide a critical isotopic baseline for coastal floodplain forests, and act as a framework for future studies of Mesozoic palaeoecology.Stable isotopes are powerful tools for elucidating ecological trends in extant vertebrate communities, though their application to Mesozoic ecosystems is complicated by a lack of extant isotope data from comparable environments/ecosystems (e.g. coastal floodplain forest environments, lacking significant C Samples were loaded into a 3.8 cm diameter sample chamber with 20 cm3 volume designed for rapid sample switching, kept under a constant flow of approximately 40 ml min−1 of ultra-high purity helium, with CO2 gas liberated through ablation travelling to a Thermo ScientificTD GasBench II, being cryogenically focused in a liquid nitrogen trap for 3 min, then heated to 25°C, passed through a Nafion water trap and then moved into a Thermo ScientificTD DeltaplusXL mass spectrometer for measurement. The sample chamber was heated and kept at approximately 50°C at all times, and background CO2 and H2O blanks were measured prior to each sample ablation session to ensure proper pre-analysis chamber and sample degassing. Calibrations were performed based on repeated measurements of NBS-18 and NBS-19, along with repeated analyses of a polished slab of an internal laboratory standard calcite (WS-1) to monitor/correct for analytical drift between sessions, as per [The laser-ablation gas-chromatography isotope-ratio-mass-spectrometry (LA-GC-IRMS) system used in this study is the same as that reported in Larson & Longstaffe , and is , as per .2 at 70°C for 60 min, then sampled through a Thermo ScientificTD Gasbench II at a column temperature of 60°C, and then measured in continuous-flow mode, along with a CO2 reference gas, using a Thermo ScientificTD MAT253 IRMS. Values of δ13C and δ18O were calculated based on calibrations performed on repeated measurements of IAEA-CO-8, NBS 19 and IAEA-CO-1 standards.Carbon and oxygen isotope compositions of bone bioapatite structural carbonate were analysed using a continuous-flow isotope-ratio-mass spectrometry system (CF-IRMS), beginning with weighed powders (approx. 1000 µg) being reacted with orthophosphoric acid to produce CO2 and NOx (later reduced to N2) through flash combustion at approximately 1700°C under oxygen atmosphere conditions. δ13C and δ15N calibrations were determined through repeated measurements of IAEA-CH-3, IAEA-CO-1, USGS-34, IAEA-NO-3 and IAEA-N-2 standards.Carbon and nitrogen isotope compositions of hair and claw keratin were analysed using a continuous-flow isotope-ratio-mass-spectrometer system (CF-IRMS) via an automated Eurovector elemental analyser (EA) coupled to a Thermo Scientific MAT253 IRMS. Clipped keratin samples were weighed (approx. 200 µg) and placed into tin cups, and then dropped individually to the EA, where the keratin is converted to COTD MAT253 IRMS in continuous-flow mode using Conflo III open split interface. Calibrations for δ18O were based on repeated measurements of USGS-42 and USGS-43 keratin standards.For oxygen isotope analysis of claw and hair keratin samples, weighed keratin clippings (approx. 180 µg) were added to silver cups, then pyrolysed using an automated HEKAtech HT-EA oxygen analyser at approximately 1400°C using helium as a carrier gas. The CO gas produced through this process was passed through an Ascarite trap, separated on a 5 Å Molsieve, and then measured using a Thermo Scientific2.6.δtissue − δdiet (or δdiet = δtissue − Δ) or Δ = δtissue − δtissue [tissue-diet were unavailable, and so average TEFs were calculated for each tissue using the literature data for related taxa with similar diets . The individual isotopic compositions for each sample prior to applying TEF values are listed in electronic supplementary material, tables S4 and S5. Bioapatite δ18O data represent either bioapatite structural carbonate oxygen or laser-produced total-oxygen (LPTO), depending on the particular sample/method. LPTO data represent a combination of phosphate + carbonate + hydroxyl oxygen, though the hydroxyl contribution is minimal per [δ18O alone (i.e. by 1.2‰ per Cerling & Sharp [a) allow bioapatite oxygen isotope data from structural carbonate and LPTO to be converted to phosphate oxygen isotope equivalents to facilitate comparison and discussion.Differences often exist between stable isotope compositions of carbon, nitrogen and oxygen as recorded in a sampled tissue, and the compositions of those same isotopes in the original diet or other intake for an organism ,2,4,8. T δtissue ,86,111.  δtissue –100,105. δtissue ,11,12, n δtissue –116. Des δtissue . Here, wimal per , leading & Sharp  and by 0 & Sharp ). Compar & Sharp ,112,118 2.7.Data analyses were performed, and base plots made, using the R programming language and contained core packages , along wCommunity-level comparisons were performed on the stable carbon, nitrogen and oxygen isotope results for all sampled species. Stable isotope data were adjusted through the application of TEFs (for nitrogen and carbon) and carbonate-phosphate/LPTO-phosphate discrimination factors (for oxygen), as described above . Comparisons were made using combined keratin (hair and claw) and bioapatite (bone and tooth) data, respectively. In each case, species isotopic ranges were plotted for keratin nitrogen, keratin carbon, bioapatite carbon and bioapatite oxygen to test for species-level differences, and illustrated in combined bi-plots of isotopic ratios  to more fully visualize the range of community isotopic distribution and possibly niche space occupation, under the methodological constraints present in a Mesozoic system . These distributions were also compared with observational natural history records compiled from the literature . These comparisons were used to assess the inferences made from the distributions themselves (nitrogen isotope results being used in somewhat the same way relative to carbon isotope or oxygen isotope results), given the general lack of availability of such data in fossil systems.et al. [δ18Osurface water = (δ18Ophosphate − 21.3 + 3h)/0.74),— faunivorous mammal (δ18Osurface water = (δ18Ophosphate − 22.7 + 3.9h)/0.64),— omnivorous mammal (δ18Osurface water = (δ18Ophosphate − 26.8 + 8.9h)/0.76),— herbivorous mammal (δ18Osurface water = 0.82δ18Ophosphate − 19.13),— reptile/fish .Body water oxygen isotope concentrations were calculated from bioapatite oxygen isotope data  for each sampled taxon, using a combination of taxon/physiology-specific equations from Kohn  , and then combines the resultant river oxygen isotope composition with the oxygen isotope data for ectothermic fish (in their case derived from fossil gar ganoine) in order to calculate river temperature. This procedure was modified to calculate δ18Oriver for each individual endothermic species using the equations noted above, with the mean of the resultant δ18Oriver values entered into the temperature equation, and with the mean δ18Ophosphate of all ectothermic taxa used in place of using a single gar taxon. Mammalian physiology/body temperature is considered in these calculations via the Kohn endotherm equations . As noted above, individual taxon temperature estimates were also calculated using species-specific or diet/physiology-specific equations where available, allowing for comparison with the results of the modified two-part endotherm–ectotherm mean temperature calculation. In all cases, calculated temperatures were also compared with water temperatures recorded hourly for the Atchafalaya River, obtained from the United States Geological Survey  [The first part uses the Kohn  mammaliaity, LA) , coverin3.3.1.See electronic supplementary material, S1 for detailed descriptions of the analytical results of all tissue comparisons and methodological testing. Isotopic results for nitrogen, carbon and oxygen all fall within broadly expected ranges based on related mammals, reptiles and fish, though the degree of analytical variability was dependent on several factors. In general, variability was low in repeated sampling of the same tissues of a given specimen, but became higher when comparing between tissues, or between specimens and species. Bulk powder isotopic analyses produced more consistent, low variability, results, whereas laser ablation isotopic analyses resulted in higher variation . This is probably due to the nature of the sampling and analytical methods, as bulk powders naturally lead to a more consistently mixed and averaged isotopic signal, whereas laser ablation, by nature of the small spatial scale of the samples, potentially reveals temporal variability in isotopic signal. This higher variability is consistent with previous stable isotope studies using laser ablation techniques ,123.Sus  and Mephitis (skunk) exhibited the greatest variation in carbon isotope compositions. Within the bioapatite samples, the greatest degree of isotopic variation was seen in aquatic taxa, particularly Atractosteus  and Lepisosteus (longnose/spotted gar). Offsets between keratin and bioapatite isotopic compositions in mammalian taxa match literature expectations . While no direct Alligator hard tissue to soft tissue comparison could be found in the literature, the keratin-bioapatite offsets in sampled Alligator match those of other known reptiles . While keratin and bioapatite carbon isotopic ranges were generally consistent in taxa sampled for both, there were moderate differences in average signal recorded in several taxa, including Neovison (mink), Lontra (otter) and Sciurus (squirrel).When comparing carbon isotopic compositions taxonomically, variation was greater in certain species when compared with others in the overall sample. Within the keratin samples, Offsets in oxygen isotopic compositions of bioapatite tissues obtained via bulk powder carbonate sampling and laser ablation total oxygen sampling for the same specimens were on average approximately 7‰, consistent with the literature expectations ,118,123 3.2.δ15N and δ13C (a), and bioapatite δ13C and δ18O (b) were produced to assess the range and variation in isotopic compositions for each taxon, and visualize resource/niche partitioning in this community in multi-dimensional space. Coloured hulls in these figures (with associated colour-filled points) indicate broad dietary/guild assignment . Keratin δ15N effectively separated groups of taxa by relative trophic position (a) over a range of approximately 0 to + 10‰, with faunivorous taxa  possessing mean δ15N > + 7.5‰, omnivorous taxa  possessing mean δ15N of +4‰ to +6.5‰, and herbivorous taxa  possessing mean δ15N < +4‰. In both keratin δ13C (a) and bioapatite δ13C (b), taxa occupied similar ranges of isotopic space (approx. –30 to –15‰ for keratin δ13C and –35 to –20‰ for bioapatite δ13C). No significant outlier taxa are present in the bioapatite δ13C results, whereas three taxa in the keratin dataset  had average δ13C that were approximately 4 to 8‰ higher than the mean of most sampled taxa. Herbivores typically had lower δ13C than most other taxa, though moderate to considerable overlap exists in δ13C among most taxa for both keratin and bioapatite. Fully aquatic faunivores (i.e. fish) had higher δ13C than most other faunivores, and little difference existed in δ13C distributions for sampled terrestrial faunivores and omnivores. A high degree of overlap also existed in bioapatite δ18O, with a total range of approximately +13 to +23‰. Within this range, omnivores and faunivores typically had the lowest δ18O, followed by fish and herbivores. The main exceptions to this pattern were the high values for a large herbivore, Odocoileus (mean δ18O of approx. +23‰), and low values of Amia (mean δ18O of approx. +15‰) when compared with other fish or semi-aquatic taxa.Mean isotopic results from bioapatite and keratin samples of each specimen, with TEFs applied, are presented in and δ13C a, and biand δ18O b were prposition a over a tin δ13C a and bioite δ13C b, taxa o3.3.et al. [Canis latrans most closely reflected mean annual river temperature (20.6 versus 20.3°C), and the majority of bioapatite-derived temperatures plotted between 10 and 15°C. This is particularly true of temperatures derived from aquatic ectotherm bioapatite, in which all sampled taxa produced estimates consistent with non-summer Atchafalaya River water temperature ranges. When the proposed multi-taxic two-part endotherm–ectotherm method  is used4.4.1.3 , or C4  photosynthetic pathway, being therefore low in δ13C relative to modern atmospheric CO2 (approx. –8.5 to –8.4‰) [15N becoming more enriched as the trophic level increases [δ18O record consumed water sources, depending on the diet/behaviour of an organism, and are often closely related to surface water conditions, local and regional patterns of temperature, precipitation, humidity, evaporation, elevation and marine versus freshwater settings [δ18O may predominate in the signal present in a given tissue. These considerations include whether an organism obtains water by drinking  or primarily through its diet , the degree of humidity dependence in a given taxon, and metabolism (i.e. ectothermic versus endothermic) of a taxon [The relative amounts of different stable isotopes preserved in the tissues of an organism depend on several factors. Stable carbon isotope ratios, particularly in terrestrial systems, generally relate to the plant source at the base of the food web, which typically follow either the Co –8.4‰) ,111,124.o –8.4‰) . Stable o –8.4‰) , physiolo –8.4‰) , forest o –8.4‰) ,127, disncreases ,111. Thincreases ,128–130.ncreases ,111,131.ncreases ,49,111. settings ,121. The a taxon ,49. Thes4.2.Comparisons of mean isotope distributions  illustraδ15N is more effective at partitioning taxa by relative trophic level , matching predictions from natural history data . Both keratin and bioapatite δ13C have considerable overlap in terms of overall distribution, among guilds, as well as separately among the sampled species (3-based systems [δ13C ranges of Neovison (mink), Mephitis (skunk) and Sus , all of which are richer in 13C relative to the community average, suggesting some degree of C4 influence on their diets. Interestingly, the mean bioapatite δ13C of Neovison is approximately –27‰, whereas its keratin δ13C is approximately –18‰, suggesting that whatever C4 influence existed on the diet of Neovison in this sample may have been present on a shorter/recent timescale , which is known to feed on a wide variety of terrestrial and aquatic organisms, shifts its diet opportunistically or seasonally between C3  and C4  feeding prey, and the C3 source happens to have been what was preserved in these individuals during the relatively short period of mammalian enamel formation early in ontogeny [The degree of overlap present, and ecological interpretation possible, in these data is dependent on the particular isotope and tissue being discussed. Keratin  species , and the systems ,111. A mo months ,132) whef a year , whereasf a year ). It is ontogeny .Despite the observed overlaps, some broad patterns can be seen in the keratin and bioapatite carbon isotope results at both the species and guild levels. Among larger grouping, such as diet/guild . Previous studies comparing differences in the carbon isotope compositions of photosynthetic versus reproductive plant tissues, however, typically find the latter  to be 13C-enriched rather than 13C-depleted [Sciurus and Neotoma is also supported by the relatively higher mean bioapatite δ18O for these taxa, as arboreality has previously been shown to correlate with 18O-enrichment [Relatively little evidence exists to suggest a widespread canopy effect contribution to the overall pattern of either the keratin or bioapatite δ13C ,127. It habitats ,136. Anodepleted , suggestrichment .Procyon and Didelphis contain overlapping isotopic ranges, though the former overlaps with multiple herbivorous taxa, whereas the latter does not (overlapping only with Sylvilagus). This difference may be the result of aquatic invertebrates/bivalves forming a large component of the diet of Procyon when compared with Didelphis , as similar diets have previously been found to be more negative in δ13C [δ13C  [Alligator, Ursus, Lontra). In the case of both Alligator and Ursus (bear), it is known that these animals can have varied diets composed of terrestrial and/or aquatic sources [Ursus there is also probably a large herbivorous component to the diet [δ13C range of Ursus structural carbonate from bone bioapatite in this study is interesting as it contrasts with recent reports [4 plants  seem to make up a large component of the overall diet. It is possible that these Ursus specimens did not feed as heavily on human crops. Given the seasonal nature of that feeding [4 effects when combined with primarily C3 feeding during the majority of the year. Crustaceans, fish  and mammals (e.g. Myocastor) are typical prey for Alligator in Louisiana [δ13C range of Alligator relative to Myocastor and sampled fish taxa. The three sampled fish taxa  have carbon isotope ranges consistent with aquatic feeding on pelagic/limnetic and littoral, rather than benthic, food sources [Lontra, being depleted of 13C relative to other aquatic taxa and highly variable (for keratin), may be the result of several factors. Primarily freshwater feeding in Lontra would explain the lower δ13C if individuals of other aquatic taxa  have a higher amount of estuarine or marine influence in their feeding [Lontra, consuming mostly fish in summer and crustaceans/bivalves in winter, is responsible for its more divergent δ13C [δ13C of bioapatite tissues in Lepisoteus and Atractosteus relates to their natural history. A study of seasonal habitat use in Lepisosteus oculatus from the Atchafalaya River (and surrounding lakes and streams) found considerable variation in home range size/shape [Among higher-level consumers, several species-level patterns exist. Omnivorous species such as  in δ13C . Relativdelphis) , though  sources ,141, andthe diet . The δ13 reports  of their feeding , it is a sources ,142,143. feeding . It is aent δ13C , as bentent δ13C ,142. Carze/shape . Variatize/shape . This vaδ13C data would be even more difficult to interpret. It is also possible that some component of the isotopic distributions of taxa sampled in these analyses may be influenced by other factors, such as species-specific ontogenetic dietary niche shifts [Resource mixing of the form suggested for gar may also account for some of the intra-species variability and overall isotopic range overlap seen in the Atchafalaya dataset. Just as this hypothesized mixing of organic carbon, sediment and even individual aquatic taxa (such as gar) may occur during annual flood-induced inundations, terrestrial taxa feeding across these landscapes may be consuming resources that are less distinct in isotopic composition than is the case in other environments. As well, living in annually flooded, often water-fragmented habitats may result in more mixed-feeding diets for taxa in these communities, deriving food resources from a mix of terrestrial and aquatic sources, rather than more specialized behaviours. An important consideration here is that without the observed natural history data available for this extant system , the patterns in these e shifts ,148, bode shifts  or physie shifts , and fur4.3.Lepisosteus sp., A. spatula, A. mississippiensis, M. coypus, L. canadensis, S. aquaticus) all being within 1°C of one another, and the oxygen isotope compositions of these taxa being similar to unpublished USGS records of Atchafalaya River oxygen isotope data  had much lower calculated temperatures. The reason for the difference in δ18O and calculated temperatures for Amia compared to other ectothermic fish such as Lepisosteus and Atractosteus is not known. It may represent a partial marine or estuarine signal in the gar taxa, as marine δ18O is typically higher than that of freshwater  [Amia provides just one example of the many taxon-specific sources of variability that may exist. Behavioural factors , this wulation)  and gar ulation) . Amia prreptiles ), proporreptiles ), whereareptiles . Bone bireptiles , though humidity ,49,111. The uncertainties and associated variability present in the calculated temperatures derived from oxygen isotopes in vertebrate bioapatite in the Atchafalaya dataset present some possible problems for the use of such methods in palaeontological contexts. This variation, as noted above, may be the result of multiple factors influencing the preserved oxygen isotope composition, and potentially interfering with the oxygen isotope–temperature relationship. This suggests that alternative methods of isotope temperature reconstruction, such as the measurement of clumped isotopes , be used3-dominated systems [Although the relatively high degree of variability and overlap seen in the Atchafalaya results may appear somewhat at odds with other vertebrate isotope ecology studies ,50,53,83 systems ,154, and systems –158. The4.4.As noted in the discussion of the isotope ecology of the ARB data themselves, one particularly important implication for interpreting isotopic data from Mesozoic systems is the degree to which ecological or dietary relationships can be interpreted in the presence of broad isotopic overlap between species. If overlap is considerable, and if there is not significant separation in the mean values between species, then drawing direct conclusions regarding the fine-scale ecological patterns present in such a system may not be possible .b and Alligator), ‘holostean’ fish  and metatherians (Didelphis), should be useful for future direct comparisons with Mesozoic systems, given the presence of many morphologically, ecologically and/or phylogenetically similar taxa in such assemblages [The relative relationships of bioapatite carbon and oxygen isotope distributions of taxa presented in this study figures b and 3, emblages ,159–162.emblages  and taxoemblages  similariδ13C and δ18O within and among enamel, bone and ganoine tissues from gar in this study, relative to other sampled taxa  systems. In several isotopic studies of Mesozoic dinosaurs, samples of gar ganoine have been used to facilitate intra- and inter-specific comparisons of herbivorous dinosaur enamel stable isotope data sampled from single sites or across formations ,23,24. Wδ15N values that did not show the expected increase with increasing trophic level , but instead exhibited a high degree of trophic level mixing . Based on our results, it seems likely that those inconsistencies are the result of methodological issues, contamination or diagenetic alteration, or because nitrogen isotopes do not preserve in fossils of such considerable age. While evidence of original organic preservation in pre-Cenozoic fossils remains controversial [Nitrogen isotope results in the Atchafalaya dataset, derived from keratin, are not particularly applicable to most pre-Cenozoic fossil stable isotope analyses due to a presumed lack of appropriate soft tissue preservation. In our view, previous research examining nitrogen isotope distributions (from high molecular weight compounds) in Mesozoic vertebrate communities  producedoversial ,163–172,oversial –175 sugget al. [et al. [A number of studies using carbon and/or oxygen isotope data have made ecological, environmental or physiological inferences about Mesozoic vertebrate communities and the taxa within them ,176,177.et al.  performeet al. ). What i [et al.  with gre [et al. ), whereaSciurus, Myocastor and Sylvilagus are approximately 2.0–4.5‰ and approximately 0.5–2.5‰ from one another, respectively (similar to hadrosaurs and ceratopsians), with broadly overlapping total isotopic ranges  and lack the confounding concern of diagenetic alteration that accompanies any investigation of ancient stable isotopes . From th5.3-dominated coastal floodplain forest system. Measured ranges of δ13C (from keratin and bioapatite tissues) and δ18O  were found to have considerable overlaps among individuals and taxa, suggesting a high degree of resource mixing, terrestrial–aquatic interchange, and a possible lack of ecological saturation in this system. Even with well-established natural history data on ecological variables for our ARB taxa, such as trophic habits and microhabitat use, the subtleties involved in identifying these patterns in our isotopic data highlight the difficulties involved in detecting and interpreting ecological patterns in a complex C3-based system. It stands to reason that for a palaeoecological system where the description of niches often requires significant inference , using isotopic data to describe fine-resolution details of niche partitioning should be done conservatively. Consequently, though carbon isotope data can provide some meaningful ecological data in these systems, we also recommend that novel stable isotope systems be considered in future investigations of trophic and niche structure in C3 ecosystems. Our proposed multi-taxic modification to the two-part endotherm–ectotherm equation provides a surprisingly accurate method of temperature estimation, and may be of considerable use in palaeoenvironmental analyses. This research provides a fundamental comparative baseline for assessing isotopic variation and testing ecological hypotheses in future studies of analogous or near-analogous Mesozoic systems. Comparisons of this nature should assist in constraining predictions and limiting the degree to which palaeoecological-proxy data may be over-interpreted.This study provides the first broad-scale vertebrate isotope ecology case study with multi-taxic, multi-tissue and multi-isotope sampling from a modern C


Antenatal depression is associated with a broad range of suboptimal outcomes in offspring, although the underlying mechanisms are not yet understood. Animal studies propose inflammation and glucocorticoids as mediators of the developmental programming effect of prenatal stress on offspring stress responses, but studies in humans are not yet at this stage. Indeed, to date no single study has examined the effects of a rigorously defined, clinically significant Major Depressive Disorder (MDD) in pregnancy on maternal antenatal inflammatory biomarkers and hypothalamic-pituitary (HPA) axis, as well as on offspring HPA axis, behavior and developmental outcomes in the first postnatal year.A prospective longitudinal design was used in 106 women  to study the effect of MDD in pregnancy and associated antenatal biology (inflammatory and cortisol biomarkers), on offspring stress response , early neurobehavior , and cognitive, language and motor development .Compared with healthy controls, women with MDD in pregnancy had raised interleukin (IL) IL-6 (effect size (δ) = 0.53, p = 0.031), IL-10 , tumor necrosis factor alpha  and vascular endothelial growth factor , together with raised diurnal cortisol secretion , raised evening cortisol , and blunted cortisol awakening response , and an 8-day shorter length of gestation . Furthermore, they had neonates with suboptimal neurobehavioral function in four out of five NBAS clusters measured (range of δ = 0.45–1.22 and p = 0.049–<0.001) and increased cortisol response to stress at one year of age . Lastly, maternal inflammatory biomarkers and cortisol levels were correlated with infant stress response, suggesting a mechanistic link.This study confirms and extends the notion that depression in pregnancy is associated with altered offspring behavior and biological stress response, and demonstrates that changes in maternal antenatal stress-related biology are associated with these infant outcomes. These eAlthough mechanisms for developmental programming in humans are likely to be complex, it is postulated that, as in animals, exposure of the fetus to high levels of inflammatory biomarkers or glucocorticoids is a key mediator, resulting in subsequent effects on HPA axis, behavior, and cognitive function of the offspring . While cOf note also is that developmental programming studies so far have only measured the associations between two variables at a time, limiting our understanding of the connections of antenatal depression with both offspring behavior and stress-related biology. For example, studies have found associations (i) between antenatal symptoms of depression and offspring HPA axis or inflammation ; or (ii)22.1In a prospective longitudinal observational study, we compared a cases group with a DSM-IV diagnosis of MDD in pregnancy (and their offspring) with a control group of healthy pregnant women (and their offspring) up to one year postnatally. Maternal socio-demographics, obstetric and physical risk factors, together with clinical status and inflammatory markers, were assessed at baseline (25 weeks gestation), and HPA axis at 32 weeks of pregnancy. Gestational age at birth and birth weight were recorded, and neonatal neurobehavioral function was assessed at 6-days postnatal. Infant cortisol reactivity (response to the pain stress of routine immunizations) and basal activity (morning and evening) was also assessed at 2- and 12-months postnatal. Finally, infant development was assessed at 12-months postnatal. Outcome measures were assessed blind to caseness. The study was approved by King’s College Hospital Research Ethics Committee, and all participants provided written informed consent.2.2The sample comprised 106 women recruited in the late second trimester of pregnancy (25 weeks gestation): 49 cases with MDD in pregnancy  and 57 healthy controls  all at King’s College Hospital. Of the 49 cases, 31 (63%) met DSM-IV criteria for MDD at 25 weeks gestation (baseline visit) and 18 (37%) were assessed to have met criteria for MDD in early pregnancy but not any longer by baseline. Of the total sample, 41 (84%) had a past history of MDD. Inclusion criteria were: women over 18 years of age with a singleton pregnancy; cases with a DSM-IV diagnosis of MDD in pregnancy; and controls without any current or past DSM-IV axis I diagnosis. Exclusion criteria were: uterine anomaly, known obstetric complications in the index pregnancy, severe or relevant chronic medical conditions, such as cardiovascular disease, metabolic or endocrine disorder, for example gestational diabetes and hypertension. Cases were excluded if presenting with any current DSM-IV diagnosis other than co-morbid anxiety disorder, if having a past history of psychosis or bipolar affective disorder, or if taking antidepressant medication at baseline.As expected in a longitudinal study of an inner city group of people, subject retention reduced over time, and at 1 year postnatal only 87 mother-infant dyads (51 controls and 36 cases) were assessed. However, there was no statistically significant difference at any time point between the proportion of cases and controls remaining in the study; furthermore, there were no statistically significant differences in socio-demographic information at baseline between those who did and did not complete the 1 year assessments, either for cases or for controls (data not shown). However, subjects who did not complete the 1-year assessment had higher BDI and STAI scores at baseline and a greater proportion who had smoked in pregnancy (see below), which may have influenced some of the findings (see Discussion).2.3All subjects were assessed for current and past DSM-IV axis I disorders at baseline using the Structured Clinical Interview for DSM-IV (SCID I – CV) . We addiThe most relevant socio-demographic and medical factors are presented in 2(1) = 9.6, p = 0.005), which may have influenced some of the findings (see Discussion).Compared with women who remained in the study, subjects who did not complete the 1 year assessment had higher BDI  and STAI  scores at baseline and a greater proportion who had smoked in pregnancy (11 (13%) v 8 (44%), χ2.4Blood was obtained between 12 pm and 3 pm at a visit early in the third trimester ; there was no statistically significant difference in gestational age at sample acquisition between cases and controls . All samples were transported to the laboratory in a cooled box and processed within 2 h of venipuncture. Aliquots of serum were immediately frozen at −80 °C pending analysis. Serum high sensitivity C-reactive protein (hsCRP) was measured using an ELISA kit supplied by PZ Cormay, Poland; the assay was analyzed in batches on the Cobas Mira (intra- and inter-assay CV were 2.96% and 3.85% respectively). Serum IL-1α, IL-1β, IL-2, IL-4, Il-6, IL-8, IL-10, TNFα, vascular endothelial growth factor (VEGF), EGF, MCP-1, and INF-γ were measured using a cytokine chip array kit supplied by Randox Laboratories, UK; the kit employs a sandwich chemiluminescent immunoassay, described in our previous work . For IL-2.5Maternal saliva samples were obtained in the third trimester ; there was no statistically significant difference in gestational age at sample acquisition between cases and controls . All subjects collected two samples, using Salivettes containing a polymer swab , at awakening and 8 pm. Cortisol awakening response (CAR) was assessed in a subset (n = 29) that also collected samples at +15 min, +30 min, and +60 min after awakening. Subjects were given a practical demonstration, verbal and written instructions, a recording log and a mechanical timer for sample collection; emphasis was placed on the accuracy of timings and procedure, and subjects were instructed not to eat, drink or smoke in the first hour after awakening or in the thirty minutes before sample collection at 8 pm. There were no differences between cases and controls, in awakening time (7:34 h ± 0:52), the time of awakening sample collection (7:39 h ± 0:52), the interval between awakening and sample acquisition (6.70 min ± 11.35) and the time of evening sample collection (20:09 h ± 0:39).Infants’ saliva samples were collected by a researcher before and 20 min after the routine immunizations at 2 and 12 months ; there was no statistically significant difference in age for infants of cases or controls at either time point . Infants’ saliva samples were also obtained on the following day, by the mother, at morning awakening and at 8 pm. Care was taken to avoid feeding for 15 min before a sample was taken. A Salivette and Salimetrics childrens swab (SCS) were used to collect saliva by the researcher on the immunization day; while, for ease of use, a Sorbette arrow was used for infant saliva collection the following day by the mother. As for mothers, a sample log was used to record timings and relevant information. There were no differences between infants of cases and controls, in awakening time (7:27 h ± 1:05 at 2 months and 7:19 ± 1:01 at 12 months), the interval between awakening and sample acquisition (15.6 min ± 21.0 at 2 months and 14.7 ± 21.0 at 12 months) and the time of evening sample collection (20:05 h ± 0:44 at 2 months and 19:43 ± 0:41 at 12 months).Salivary samples were frozen at −20 °C pending analysis. Saliva cortisol was measured using a high sensitivity salivary cortisol enzyme immunoassay kit supplied by Salimetrics Europe Ltd, UK. Samples were assayed in duplicate where an adequate volume of saliva allowed. The inter-and intra-assay CV ranged from 8 to 11% and 6 to 10% respectively. The formula for the area of a trapezoid  was used2.6The Neonatal Behavioral Assessment Scale (NBAS)  was used2.72) test of the independence of variables was used for the analysis of categorical data. Pearson’s correlation (rp) was used for the analysis of association between parametric continuous variables, and Spearman’s correlation (rs) was applied to non-parametric continuous variables. In univariate analyses, group comparisons of continuous data were made using the independent samples t-test. For non-parametric data, the Mann-Whitney test was used and the z score reported. Univariate analyses that showed significant differences between patients and controls were repeated after adjustment for the IMD and smoking in pregnancy if appropriate. Cohen’s δ was calculated to estimate the effect size for group differences, or effect size was expressed by partial eta squared (ηp2) where ANOVA and ANCOVA were applied. Family-wise adjustment for multiple comparisons was used to identify the strongest findings. Mean and standard error of the mean are presented in graphs.The statistical analyses were performed in SPSS Statistics Version 21 . The analysis plan comprised cross-sectional group comparisons of maternal antenatal biomarkers, birth outcomes, neonatal neurobehavior, infant HPA axis, and development in 1-year-olds, as well as the associations between infant factors and maternal biomarkers. For all statistical tests, the data were first examined to ensure that the assumptions of the General Linear Model (GLM) were met. In order to reduce bias, data were winsorized or log-transformed prior to analyses or the bootstrap method (with 1000 samples) was employed; raw data are presented in the figures and tables. Pearson’s chi-square  = −2.45, p = 0.019). These secondary analyses confirm the notion that depressed pregnant women maintain a pro-inflammatory signature at the end of the second trimester even if their depression has improved by then.Finally, as documented in section 3.2(42.8) = −2.9, p = 0.006, δ = 0.89; p = 0.001 after adjustment for IMD) ((44.1) = −1.8, p = 0.07, δ = 0.54; p = 0. 027 after adjustment for IMD), and significantly higher evening cortisol levels (t(81) = −2.9, p = 0.004, δ = 0.64; p = 0.006 after adjustment for IMD). Lastly, depressed women had a statistically significant smaller (blunted) CAR  = 2.4, p = 0.020, δ = 0.70; p = 0.016 after adjustment for IMD)  , Panel Afor IMD) , Panel BPotential confounders did not affect these results. Smoking was not associated with these cortisol measures. Again, although pre-pregnancy BMI was not statistically different between cases and controls, we explored its possible association with the HPA axis measures; it was negatively associated with CAR only; inclusion of BMI in the covariate model for CAR did not change the findings. Diurnal and evening cortisol remained statistically significant even after adjusting for multiple comparisons. Finally, t-tests revealed no statistically significant difference in the HPA axis measures between cases who did and did not meet criteria for MDD at the baseline visit .3.3(69) = 2.9, p = 0.005, δ = 0.70; p = 0.012 after adjustment for IMD), and smoking was not associated with this measure. There was no statistically significant difference between cases and controls in any other obstetric outcome or obstetric detail .Gestational age at birth for women with spontaneous onset of labor was lower in babies of cases compared with those of controls, by approximately 8 days  of full-term babies conducted at 6 days postnatal showed that babies exposed to antenatal depression demonstrated poorer performance on all five clusters: autonomic stability (δ = 0.85), regulation of state (δ = 0.61), range of state (δ = 0.53), orientation (δ = 1.22) and motor (δ = 0.45) .Table 3AThe analyses were adjusted for IMD and other potential confounders  that differed between cases and controls, including gestational age at birth and smoking in pregnancy. For all the NBAS clusters, except ‘range of state,’ the effect of depression remained significant after adjustment for IMD and these other potential confounders . For ‘range of state’ there was a significant effect of smoking in pregnancy (p = 0.030) and the effect of depression was no longer significant after adjusting for this variable. Autonomic stability, regulation of state and orientation remained statistically significant even after adjusting for multiple comparisons.3.5Cortisol function of the two-month-old infants was assessed by measuring cortisol before and 20 min after immunizations, as well as morning and evening levels. = 27.0, p < 0.001, ηp2 = .25), that is, cortisol increased following immunization in both depression-exposed and non-exposed infants. However, there was no interaction between caseness and time  = 0.4, p = 0.51, ηp2 = .01) and no between-subjects effects  = 0.6, p = 0.45, ηp2 = .01). Thus, the magnitude of the cortisol response did not differ between those infants who had and had not been exposed to antenatal depression.For cortisol reactivity to stress , Panel ABasal HPA axis activity in the eight-week-old infants was assessed the day after their immunization by measuring awakening and evening cortisol . There was no statistically significant difference in either measure between the exposed and non-exposed infants  , Panel B3.6Cortisol function of the 12-month-old infants was assessed, as at 2 months, by measuring cortisol before and 20 min after the immunizations, as well as morning and evening levels. = 0.5, p = 0.002, ηp2 = .13), as well as a statistical trend for an interaction between caseness and time  = 3.4, p = 0.07, ηp2 = 0.05), but no within-subjects effect  = 1.9, p = 0.17, ηp2 = 0.03). These statistical findings were driven by an increase in cortisol levels following stress in the infants exposed to antenatal depression, but not in the non-exposed infants ((72) = −3.7, p < 0.001, δ = 0.87) but not before (t(72) = −1.6, p = 0.11, δ = 0.38). The mixed design ANOVA used to measure the cortisol response to stress takes account of the multiple testing, and thus this group difference is statistically robust.For cortisol reactivity to stress , Panel C infants , Panel C(29.4) = −3.4, p = 0.002, δ = 1.25) and as total postnatal burden  = −4.6, p < 0.001, δ = 1.60). Only average postnatal symptoms of depression were associated with the infant cortisol, therefore average postnatal BDI and IMD were included in the ANOVAS. The between-subjects effect of caseness in the mixed design ANOVA remained significant (p = 0.024), as did the difference between exposed and non-exposed infants in the post-immunization cortisol (p = 0.021). Thus, the increase in cortisol levels following stress in the infants exposed to antenatal depression was not driven by the postnatal depressive symptoms or other potential confounding factors.Given the well-established continuity between maternal antenatal and postnatal depressive symptoms , in addi(62) = −2.4, p = 0.020, δ = 0.61), but not morning cortisol, (t(62) = −0.7, p = 0.49, δ = 0.18 . Infants exposed to antenatal depression had higher evening cortisol at 12 months  = 1.5, p = 0.11), language  = 1.4, p = 0.16) or motor  = −0.9, p = 0.38) development at 12 months of age, as measured by BSID. The findings are the same both for the entire sample and solely for full-term infants.Compared with infants of healthy women (n = 50), those exposed to depression in utero (n = 35) did not differ in cognitive  and the infant measures that differed between cases and controls and were uniquely explained by the antenatal exposure  .Table 4CThere were no correlations between maternal antenatal biomarkers and gestational age at birth. However, there were statistically significant correlations between maternal biomarkers  and the NBAS scores of full-term babies . These correlations were all in the expected direction, i.e., elevated biomarker levels correlated with less optimal neurobehavioral function. There were also statistically significant correlations between maternal biomarkers  and infant cortisol after the immunizations at 12 months . Again, these correlations were all in the expected directions, i.e., elevated maternal antenatal biomarker levels correlated with higher infant cortisol. Taken all together, these correlations support the notion that neonatal neurobehavioral function at 6 days and cortisol stress response at 12 months are an embedded biological programming effect that is associated with the antenatal biological changes found in women with MDD in pregnancy.4We used a prospective longitudinal design and demonstrated that women with clinically-significant antenatal MDD have: (i) abnormal stress-related biology (inflammation and cortisol) in pregnancy; (ii) shorter length of gestation; (iii) neonates with sub-optimal neurobehavioral function; and (iv) one-year-old infants with increased cortisol reactivity to stress. Our findings are in keeping with the hypothesis that maternal antenatal MDD has a programming effect on offspring neurobehavioral function and HPA axis activity, and, furthermore, that maternal antenatal inflammation and cortisol are involved in the mechanistic pathway of these programming effects.4.1rd trimester inflammatory markers , HPA axis overactivity as indexed by raised evening cortisol and increased total daily cortisol secretion, and a blunted cortisol awakening response. Our findings are broadly consistent with previous literature, although the findings from previous studies of antenatal depression and inflammation or HPA axis appear mixed, and they are difficult to compare as they use different definitions or measures of depression and different measurements of inflammation or HPA axis, at different stages of gestation, different times of day and in different tissues. The most-studied inflammatory markers are CRP, IL-1b, IL-6 and TNFα, which are all more commonly raised in relation to increased depressive symptomatology  and evening cortisol. These correlations are in the same direction as hypothesized, i.e. women with antenatal MDD have higher IL-10, TNFα, VEGF and evening cortisol in pregnancy, and their infants have higher cortisol after their immunizations. This association supports the hypothesis of a mechanistic link between the antenatal biological environment and offspring cortisol stress regulation. Moreover, these findings extend those of other studies in humans that demonstrate an association between maternal antenatal cortisol , or amni4.4Compared with neonates of healthy women, those exposed to MDD in utero show suboptimal neurobehavioral function at 6 days, indexed by NBAS examination; however, there is no group difference in cognitive, language or motor development at 12-months of age. The findings in neonates mirror previous studies of antenatal depression and offspring neurobehavioral function, as described in the introduction , and furAs mentioned above, some NBAS clusters are significantly correlated with maternal antenatal inflammatory and HPA axis markers. These correlations are in the same direction as hypothesized, i.e., elevated maternal antenatal stress-related markers are associated with less optimal NBAS scores, again supporting the hypothesis of a mechanistic link for these neurobehavioral outcomes. Other studies have demonstrated associations between maternal antenatal inflammation and offspring behavior; for example, a study has shown that IL-6 is associated with impulse control in 2-year-old offspring . MoreoveThe lack of effect of antenatal depression on developmental outcomes at 12 months is potentially surprising. However, there are limited data in humans on the effects of depression in pregnancy on offspring developmental outcome, and results are conflicting . Some la4.5Despite a number of important strengths, including the prospective design, diagnostic assessment by semi-structured interview, and a broad assessment of inflammation and HPA axis markers in women and offspring over time, there are some limitations. First, subjects were drawn from a population with broad diversity in socio-economic status and ethnicity; although we have controlled for social deprivation (IMD), the numbers were too small to complete any analyses in specific social or ethnic subgroups. Furthermore, subjects were medically healthier compared with a general pregnant population, due to the exclusion criteria for the study, and indeed their pre-pregnancy BMI was not associated with depression. Second, the majority of cases had a previous history of depression or of other (non-psychotic) mental disorders, and we cannot exclude a genetic component linking maternal psychopathology with offspring biological or behavioral changes; nevertheless, we have been able to show that our main finding – the increase offspring cortisol response - is driven by depression in pregnancy and not by depression in the postpartum, thus indicating that specific effects are present for depression in pregnancy, over and above any generic risk associated with depression at other times. Third, although cases were free from antidepressant medication at baseline, 8 subjects took antidepressants later in pregnancy. These effects could not be quantified because of the infrequency of this potential confounding factor; however, exclusion of these eights subjects from the analyses did not change the findings (data not shown). Forth, principally because of difficulties recruiting such an unwell group of pregnant women, the sample size is relatively small. This limitation is compounded by sample attrition over the 15 months of study, and those who dropped out had higher BDI and STAI scores at baseline; thus, perhaps understandably, those who had more severe psychopathology early in pregnancy then seem to have struggled to remain involved in the study. Overall, these findings do not detract from our main biological finding in infants (the increased cortisol response at 12 months), as these new data show that this effect is present also in the potentially less severe group of women and their infants who had the follow-up visit. However, as mentioned above, it is possible that the lack of difference in the BSID scores may be partly due to the analyses being limited in this less severe sample. Fifth, undoubtedly, the study’s impact comes from the coexisting measures of both biological and clinical variables from both mothers and infants, over pregnancy and the first postnatal year. However, this brings the potential confounder of multiple comparisons. We have therefore conducted family-wise adjustment, and identified that the differences in maternal TNFα and diurnal and evening cortisol levels, and in infants’ autonomic stability, regulation of state, orientation as well as cortisol response to stress at 12 months, as the most statistically robust findings. Lastly, it is essential to emphasize that the proposed causative biological pathways, linking maternal cortisol and inflammatory biomarkers with infants’ NBAS scores and cortisol response to stress, are only supported by correlational analyses. Although these correlations are all in the expected direction, i.e., elevated maternal biomarker levels correlating with less optimal neurobehavioral function and higher cortisol response in infants, only testing for mediation would be able to unequivocally confirm these causal pathways. This, however, is inadvisable in our study due to the limited sample size. As such, these associations should considered at best suggestive, and requiring further validation and replication in larger samples.In conclusion the current study has addressed an existing gap in the literature regarding the possible programming effects of operationally defined, clinically significant antenatal depression, and represents the first step towards understanding the convergent inflammation- and cortisol-related mechanisms for developmental programming in humans. Studying the effects of depression at a disease level ensures that stress is meaningful and produces clinically significant impairment. Most importantly, antenatal depression is common  Biomedical Research Centre at the South London and Maudsley NHS Foundation Trust and King’s College London ; the UK Medical Research Council (MR/L014815/1 and MR/J002739/1) (CMP); The Lullaby Trust (formerly known as the Foundation for the Study of Infant Deaths) (263) ; the Psychiatry Research Trust ; the Brain and Behavior Research Foundation (SO); and Singapore Health Services Health Manpower Development Plan Award (2012) (TEC). The funding sources played no role in the study design, writing, or analysis of the paper and results.The work was supported by the following grants: The CMP has received research funding from pharmaceutical companies interested in the development of new antidepressants, such as Johnson & Johnson and Eleusis Ltd, but this project is unrelated to this funding; there are no further declarations of interest.


Toy (Condition 1) and also smiled more towards a communicative over a non-communicative person (Condition 2). However, all groups, except the HR-ASD group, selectively oriented towards a person addressing the child in different ways over an invariant social interaction (Condition 3). These findings suggest that social interaction is intrinsically rewarding for individuals with ASD, but the extent to which it is sought may be modulated by the specific variability of naturalistic social interaction.Several accounts have been proposed to explain difficulties with social interaction in autism spectrum disorder (ASD), amongst which atypical social orienting, decreased social motivation or difficulties with understanding the regularities driving social interaction. This study uses gaze-contingent eye-tracking to tease apart these accounts by measuring reward related behaviours in response to different social videos. Toddlers at high or low familial risk for ASD took part in this study at age 2 and were categorised at age 3 as low risk controls (LR), high-risk with no ASD diagnosis (HR-no ASD), or with a diagnosis of ASD (HR-ASD). When the on-demand social interaction was predictable, all groups, including the HR-ASD group, looked longer and smiled more towards a person greeting them compared to a mechanical  Social research . Social research . Accordiresearch , approacresearch  or talkiresearch . More reresearch ; low preresearch ). One stresearch . Howeverresearch . The decTo date, there is still little convergence within the findings to confidently support one hypothesis over another. This is partly due to the fact that many investigations into the origin of social interaction atypicalities were carried out in older children or adults. The profile of impairment in adulthood is likely to reflect idiosyncratic compensatory strategies or compounding effects resulting from a lifelong experiencing challenging social exchanges . DifficuBecause social signals are increasingly considered to induce similar responses as other reward stimuli do, i.e. motivational approach as well as hedonic response , new expIn the current work, we build on the above studies to test different accounts of atypical social interaction in ASD. An interactive eye-tracking task was used to examine whether toddlers with and without ASD engaged with and appreciated different types of simulated social interaction. Participants in this study were toddlers at high-risk for ASD due to having an older sibling with the disorder. Low-risk participants had no first-degree relative ASD. About 20% of high-risk participants go on to develop ASD themselves . AnotherFace vs. Toy), toddlers could choose between a social stimulus (a person greeting and smiling) and a non-social stimulus . In contrast to the paradigm used by Towards vs. Away) contrasted two social stimuli that, when looked at, displayed either a person turning and smiling towards the participant or a person turning away from the participant. According to the social motivation theory of autism, typically developing toddlers but not toddlers with ASD should preferably orient towards the more engaging social stimulus (Invariant vs. Variable) manipulated the variability of the social response received: an invariant interaction (showing the same clip in which a person addresses the child with Hello) was contrasted with a variable social stimulus . According to the hypo-priors account, toddlers with ASD should show a preference for the invariant interaction .Participants in this study were toddlers with or without familial risk for ASD, a proportion of whom were later diagnosed with ASD at age 3. 116 High-Risk (HR) participants  who had at least one older sibling with a community clinical diagnosis of ASD and 27 Low-risk (LR) participants  who had no first-degree relative with ASD enrolled in the study. All HR and LR children were full term infants  recruited from a volunteer database at the Birkbeck Centre for Brain and Cognitive Development. Families attended four lab visits at 9, 15, 27 and 36 months. The experimental data reported here has been collected on a subset of these children during the 27-month visit and the clinical diagnosis was obtained during the 36-month visit , ADI-R , SCQ Ru), adapti2.2The gaze-contingent tasks were created using 2.3(Face vs. Toy), one stimulus consisting of the video of a woman’s head, showing her profile and then turning towards the participant until facing the camera, at which point the addressed the participants saying ‘hello’ with a smile (there-after stimulus Face). This was contrasted with a second stimulus which was a video of a metallic Toy with brightly coloured arms rotating and accompanied by a light music of four tones (both video clips were 3.3-s-long). In the second and third conditions (Towards vs. Away and Invariant vs. Variable), the two stimuli consisted of the videos of two different women. In the second condition, a woman turned her head towards the participant and smiled (there-after stimulus Towards) while the other woman turned her head away from the participant until the back of her head was visible (there-after stimulus Away) (both video clips were 3.3-s-long). In the third condition, a woman always turned her head towards the participants and said ‘hello’ (there-after stimulus Invariant) while another woman turned her head towards the participant and either smile silently (1.6-s-long), said ‘hello’ (2.5-s-long) or said ‘good job’ (2.9-s-long) (there-after stimulus Variable). Only the women’s heads were visible. Different actresses were used in each condition , the first two trials of each block consisted in the presentation of the Face and Toy stimuli (the order of presentation was randomised in each block). In condition 2 (Towards vs. Away), the first two trials of each block consisted of the presentation of the Towards and the Away stimuli (the order of presentation was randomised in each block). For both conditions 1 and 2, the remaining 6 trials of each block were gaze-contingent and the videos of the stimuli were displayed with a delay of 500 ms upon the participant’s gaze being detected on one of the two stimuli. In condition 3 (Invariant vs. Variable), the second and fourth trials were not gaze-contingent and consisted of the automatic presentation of the Variable stimuli, all other trials were gaze-contingent. Each block of the condition 3 contained 17 trials. To further increase the variability of the Variable stimulus, the video clip was displayed after a random delay of 0–1000 ms while the Invariant stimulus was always presented after a 500 ms delay.The task consisted of the presentation of two dynamic stimuli displayed on the left and right side of the screen (350 × 400 pixel each) in the three different conditions. In each condition, a trial started with a central fixation , which disappeared when gaze was detected within an area covering it. The first stimulus screen depicted still images of the two stimuli. When the participants gazed at one of the two stimuli for 100 ms, this triggered the video sequence of the corresponding stimulus. For all the three conditions, only one video of the two stimuli was triggered and displayed in each trial. If the participant shifted his/her gaze towards the second stimulus, this did not trigger the corresponding video clip. Thus, the length of each trial was determined by the amount of time the participant would take to gaze to one of the two stimuli plus the length of the corresponding video. Finally, if the participant did not look at any of the two stimuli presented within 5 s from the beginning of the trial, the trial was terminated , and a new fixation stimulus was presented. To familiarize the participant with the different available stimuli, the first trials were not gaze-contingent and consisted of the automatic presentation of the video of the two stimuli. In condition 1 2.5Face from the condition 1 was presented on the left side in the first block; the stimulus Towards from the condition 2 was presented on the right side in the first block; finally, the stimulus Invariant from the condition 3 was presented on the left side in the first block). All the participants were given the same order of presentation: condition1-block1, condition1-block2, [other tasks], condition3-block1, condition2-block1, [other tasks], condition2-block2, condition3-block2.Infants were seated in their caregiver’s lap, at approximately 60 cm from the screen. The task, was embedded in a longer eye-tracking testing session. Each condition consisted of 2 blocks to counterbalance the left or right location of the two types of stimuli on the screen  and smile (code 1) were re-coded as invalid/missing data. Data from 13 participants (11%) of participants was double coded by one of the authors (TG). There was good agreement between the coders .We coded two key variables to assess the assignment of reward value to the stimuli by the participants. Firstly, we coded an eye-tracking measure, children , as an ichildren  or keybochildren  has been it more . Secondlresponse , or whetresponse . Interesresponse  as well response . The frelooking time and second looks) were computed. The looking time was the cumulative time the participants spent looking at the two different stimuli. The second looks corresponded to the participants' reorienting of gaze towards the opposite stimulus within two seconds after the initial looks. The second looks are a complementary index of reward-seeking behaviour, and more reorienting from a less rewarding stimulus to a more rewarding stimulus was predicted, mirroring the initial looks. Since the analysis of second looks were similar to the analyses of initial looks, the results of this analysis are reported in the SOM.Two additional measures  analyses that were built with a binomial distribution, a logit link function, an unstructured correlation matrix and a robust estimator. Bonferroni corrections were applied to the post-hoc analyses. Preliminary analysis revealed a strong leftward side bias in the initial looks measure (see SOM), therefore only the participants contributing valid trials from both blocks in each condition were included in the analyses .Additional analysis accounted for the fact that some of the participants were taking part in an intervention programme, involving video-mediated parental training . Althouginitial looks and looking time between the different Outcome groups, in either condition  = 0.66, p = 0.519; Condition 2: F  = 0.94, p = 0.394; Condition 3: F  = 0.04, p = 0.958, smiles between the different Outcome groups, in the Condition 2  = 0.01, p = 0.995) and Condition 3  = 0.12, p = 0.888, Condition 1, the LR group had a smaller number of valid trials than the HR-no ASD and HR-ASD groups  = 8.66, p < 0.001). Nevertheless, no significant correlation between the proportion of smiles and the average number of valid trials was found in the Condition 1 .The first 2 trials in Condition 1 and 2 (non-contingent) and the first 4 trials in Condition 3, were not included in analyses. There were no significant differences in the number of valid trials  included in the analyses of 33.13.1.1Face (condition 1), Towards (condition 2) and Invariant (condition 3), the proportion of initial looks towards these stimuli, averaged across the two blocks were entered in one sample t-tests against chance level, for each condition separately (Face significantly above chance level (t (13) = 1.74, p = 0.105, d = 0.47). In Condition 2, none of the groups preferably gazed at the stimulus Towards . Finally, in Condition 3, in contrast to the LR (t (20) = 2.57, p = 0.018, d = 0.56) and HR-no ASD (t (66) = 4.18, p < 0.001, d = 0.51), the HR-ASD group did not preferably gaze at the Variable stimulus (t (13) = 0.68, p = 0.507, d = 0.18).To assess whether the participants preferentially oriented towards the stimuli parately . In Cond3.1.2initial looks were entered in a GEE analysis. The stimuli Face (Condition 1), Towards (Condition 2) and Variable (Condition 3) were coded as 1 while the other corresponding choices were coded as 0. Trials, Block were entered in the model as within-subject factors and Outcome  as a between-subject factor. The analyses revealed a main effect of Outcome  = 24.23, p < 001 with HR-ASD making more pro-social choices than the LR (p = 0.001) and HR-no ASD (p < 0.001). There was also a main effect of Condition  = 35.63, p < 0.001, with Condition 2 receiving the least pro-social choices, significantly less than Condition 1 and 3 (both p < 0.001). There was no significant difference between these last two conditions (p = 0.285). Finally, two significant 2-way interactions between Outcome and Condition  = 15.16, p = 0.004) and Block and Condition  = 26.60, p < 0.001) were found as well as a marginal 3-way interaction between Outcome, Condition and Block  = 9.21, p = 0.056). We followed-up on this interaction with three GEEs, one for each condition. Main effects of Outcome were reported first and follow-up analyses of main effects of Block and of 2-way interaction between Outcome and Block are presented last, as there were not the main effects of interest. Condition 1: Face vs. Toy. The analyses revealed no significant effect of Outcome  = 0.06, p = 973). The analyses also revealed significant effects of Trials  = 10.17, p = 0.001) and Block  = 42.75, p < 0.001) as well as an interaction between Outcome and Block  = 6.05, p = 0.048), driven by a decrease in initial looks from Block 1–2 for the LR (p = 0.001) and HR-no ASD (p < 0.001) groups but not for the HR-ASD group (p = 1.000). Condition 2: Towards vs. Away. The analyses revealed a significant effect of Outcome  = 6.07, p = 0.048) which was driven by a higher proportion of initial looks directed to the Towards stimulus for the HR-ASD group than for the LR group (p=0.038). Significant effects of Trials  = 6.82, p = 0.009) and Block  = 29.68, p < 0.001), were also observed, but there was no significant interaction between Outcome and Block  = 1.05, p = 0.591). Condition 3: Variable vs. Invariant. The analyses revealed a non-significant main effect of Outcome  = 0.91, p = 0.634). There was no significant main effect of Trials  = 1.54, p = 0.214) but a significant effect of Block  = 22.32, p < 0.001) as well as a significant interaction between Outcome and Block  = 8.12, p = 0.017) which was driven by a higher proportion of prosocial choices for the LR group compared to the HR-ASD group in the first block (p = 0.010) and by a decrease in initial looks from Block 1–2 for the HR-no ASD group (p < 0.001).To evaluate the participants’ visual orientation towards one of the two stimuli of each condition over the course of the task, the 3.23.2.1target stimuli Face (Condition 1), Towards (Condition 2) or Variable (Condition 3) compared to the non-target stimuli Toy (Condition 1), Away (Condition 2) or Invariant (Condition 3) were computed as follow: “looking time towards target/(looking time towards target + looking time towards non-target)”. The proportion of looking time were then entered in one sample t-tests against chance level of 0.5, for each condition separately . Condition 2: Towards vs. Away. None of the groups spend more time looking at either of the Towards or Away stimuli . Condition 3: Variable vs. Invariant. Both the LR and the HR-no ASD groups looked longer towards the stimulus Variable compared to the Invariant stimulus . This was not the case for the HR-ASD group (t (13) = 0.72, p = 0.484, d = 0.19). Overall, the measure of initial looks and the measure of looking time seem to reflect the participants’ first selection. The participants spent more time looking at the stimulus their first gazed at. Indeed, these two measures were found to be highly correlated in all three conditions (Condition 1: r (115) = 0.94, p < 0.001; Condition 2: r (101) = 0.78, p < 0.001; Condition 3: r (102) = 0.94, p < 0.001).The proportions of looking time towards the parately . Conditi3.2.2Face vs. Toy, Towards vs. Away, Variable vs. Invariant) as within-subject factor was also conducted to examine any differences of looking time between the three conditions. No main effect of Outcome nor interaction between Outcome and Condition were found . However, a main effect of Condition was found  = 19.83, p < 0.001, ηp2 = 15). This effect was driven by a higher proportion of looking time towards the Face (vs. Toy in Condition 1), compared to the stimulus Towards  and the stimulus Variable . No differences in proportion of looking time towards the prosocial stimuli  between the Conditions 2 and 3 were found (p = 0.296).A mixed ANOVA examining the proportion of looking time with the group Outcome as between-subject factor and the factor Condition  due to the difficulty to code facial expression of the participants in the video recordings or due to missing video recordings. A description of the characteristics of this sample is reported in the SOM to show that they were representative of the whole group. Condition, Trials and Block were entered as within-subject factors and Outcome as between subject factor in the model. The Type of stimulus that was triggered on each trial  was also included as a within-subject factor in the model. This analysis yielded a significant effect of Condition  = 57.11, p < 0.001), with most smiles in Condition 1 and least smiles in Condition 2  and a marginal effect of Outcome  = 5.54, p = 0.063). There was also a main effect of the Type of stimulus, with the prosocial stimuli  eliciting more smiles  = 39.07, p < 0.001). Finally, two significant interactions between Outcome and Condition  = 33.06, p < 0.001) and between Condition and Type of stimulus  = 89.17, p < 0.001) were found. We followed-up on the significant interaction between Outcome and Condition with three GEEs, one per condition. Condition 1: Face vs. Toy. The analyses revealed no significant effect of Outcome  = 5.15, p = 0.076) but a significant effect of Type of stimulus, showing more smiles towards the stimulus Face than the stimulus Toy  = 58.97, p < 0.001). There was also a significant 2-way interaction between Outcome and Type of stimulus  = 14.05, p = 0.001). When examining the proportions of smiles separately for the stimuli Face and Toy, this yielded an effect of Outcome for the stimulus Face  = 6.24, p = 0.04), driven by a higher proportion of smiles from the HR-ASD group than the HR-no ASD group (p = 0.015). Outcome groups did not differ in the proportion of Toy choice trials eliciting smiling  = 4.73, p = 0.09). There were no significant effects of Block  = 2.28, p = 0.131) or Trials  = 0.06, p = 0.814). Condition 2: Towards vs. Away. The analyses revealed a marginal effect of Outcome  = 5.80, p = 0.055). The HR-ASD group smiled less overall than the HR-no ASD group (p = 0.008). There was also a significant main effect of Type of stimulus which was due to the participants smiling more in response to the stimulus Towards than the stimulus Away  = 23.72, p < 0.001). Two way interactions could not be computed due to quasi-complete separation, which reflects both the small sample going into each group and the very low rates of smiling in this condition, with HR-ASD all scoring zero when the Away stimulus was triggered. No significant effects of Trials  = 1.47, p = 0.225) or Block  = 1.00, p = 0.316) were found. Condition 3: Variable vs. Invariant. The analyses revealed a significant effect of Outcome  = 7.15, p = 0.028). Post hoc analyses revealed that on the whole, the HR-ASD smiled significantly more than the HR-no ASD group (p = 0.008). There was no effect of Type of stimulus  = 0.08, p = 0.784), no significant 2-way interaction between Outcome and Type of stimulus  = 2.30, p = 0.316). There was no effect of Trials  = 1.13, p = 0.288) but a main effect of Block  = 4.76, p = 0.029).To evaluate the participants’ hedonic response towards the stimuli of each condition, positive facial expressions categorised as analysis . A small4Young children with ASD encounter difficulties in interacting in social contexts from early on, and this aspect is a central characteristic used in the diagnosis of ASD . Recent 4.1initial looks and looking time were observed for the Face (vs. Toy) and Variable (vs. Invariant) stimuli but to a lesser extent for the stimulus Towards (vs. Away). A preference for orienting and maintaining attention to the social stimulus Face over the Toy was observed in typically developing toddlers (LR-group). Attentional biases to orient towards face-like stimuli could explain such preference since the two stimuli were visually dissimilar from the onset of the task (Face (vs. Toy) increased over the course of the task. This preferential orienting was supported with the analyses of second looks (see SOM) showing that typical toddlers also shifted more their attention towards the Face after an initial gaze at a very salient spinning Toy than the reverse. Secondly, toddlers also smiled more towards this stimulus compared to the Toy, suggesting that the Face is assigned positive hedonic value , unlike the social stimuli in the other two conditions to which the toddlers were exposed. Indeed, in another study, infants look longer to faces with infant directed speech than silent faces  participants. A decrease in gazing at the stimulus nt faces . The AwaVariable interaction was preferably looked at over the Invariant stimulus and preferences for the Variable stimulus remained stable throughout the task suggesting that the four initial trials were sufficient to induce preferential orienting in typically developing toddlers (LR group). In the absence of a control condition contrasting Invariant and Variable non-social stimuli, these biases towards the Variable stimulation cannot be assumed to be specific to social stimuli. However, the social Invariant stimulus was very similar to the social stimulus (Face) used in the first condition, which implies a hierarchy of preferences in which a Variable social stimulus elicits preferential orienting over an Invariant social stimulus, in turn preferred to a non-social stimulus. At the same time, the Variable stimulus did not elicit more frequent smiles; thus, the preferential orienting to the Variable stimulus might not be driven by stronger hedonic value per se.In the third condition, the Across all conditions, this task appears to successfully capture social reward in populations that cannot express preferences verbally or using manual choices. Moreover, we succeed at characterising both incentive and hedonic aspects of reward, thus providing a fuller picture of toddler’s engagement with social stimulation.4.2initial looks to the Face than the Toy stimuli, the GEE analysis comparing the different Outcome groups failed to show significant differences in the initial looks. Moreover, just like the other groups, HR-ASD showed longer looking time and more frequent second looks towards the Face stimulus (see SOM). HR-ASD also smiled more towards the Face stimulus than the Toy and smiled more towards the Face than the other at-risk group did. Thus, the current study contrasts with previous findings, in which toddlers with ASD showed more preferential looking towards geometric patterns as opposed to dynamic social scenes  and could have led to a reduced overall hedonic response for the HR-ASD group. We should note, however, that the HR-ASD smiles less in response to the more impoverished social response, in Condition 2, than the other groups, but more in response to being greeted by Hello, in Condition 1. Future studies will have to determine whether this reflects the higher familiarity with the later stimulus.In Condition 2, as mentioned above, despite not triggering preferential orienting, the stimulus initial looks and longer looking to the stimulus Variable, the HR-ASD group did not show a preference for either stimulus. We had initially predicted that HR-ASD would possibly prefer the Invariant stimulus. A preference for variable but relatively simple events in typical development is supported by previous studies showing that typically developing infants preferably orient towards neither too predictable or unpredictable, and neither too simple or too complex stimuli . A better controlled study would have compared the current Variable condition with 3 Invariant conditions, each depicting one of the stimulus types used in the Variable condition. Follow-up studies will have to address these confounding factors in search for more solid evidence for social interaction difficulties in ASD stemming from a preference for more predictable interaction.When the social stimulus was less predictable, in the third condition, contrary to all the other groups who showed significantly more  stimuli . This su stimuli . We offeOur evidence for typical social orienting and emotional engagement with social stimuli is in line with other studies on younger populations at risk e.g. . Some st5In the current study, toddlers with ASD made initial saccade, reoriented their gaze, and smiled more towards a person addressing them than towards a toy. A typical reward-seeking behaviour and hedonic response towards social stimuli seem to be present in toddlers with ASD which goes against theories suggesting impaired social orienting or impaired motivation to engage with social stimuli. However, when presented with variable and invariant social stimuli, low-risk control and high-risk toddlers, who do not have ASD, selectively oriented their attention towards the variable interaction, while toddlers with ASD showed no preference for either stimulus. This lesser drive towards variable stimulation may reflect either a shift in the bias towards more predictable information or general difficulty with processing event statistics. To our knowledge, this is the first study assessing simultaneously the effect of the rewarding and variable nature of social stimuli in toddlers with and without ASD. This was made possible by using gaze-contingent eye-tracking, which allows the interaction to be controlled by the participants. This method will allow the finer manipulation of simulated social interaction, to further probe the condition under which impaired social interaction emerges in ASD.None.


Experimental autoimmune encephalomyelitis (EAE) is attenuated in nicotinic acetylcholine receptor (nAChR) α9 subunit knock-out (α9 KO) mice. However, protection is incomplete, raising questions about roles for related, nAChR α10 subunits in ionotropic or recently-revealed metabotropic contributions to effects. Here, we demonstrate reduced EAE severity and delayed onset of disease signs in nAChR α9/α10 subunit double knock-out (DKO) animals relative to effects in wild-type (WT) control mice. These effects are indistinguishable from contemporaneously-observed effects in nicotine-treated WT or in α9 KO mice. Immune cell infiltration into the spinal cord and brain, reactive oxygen species levels in vivo, and demyelination, mostly in the spinal cord, are reduced in DKO mice. Disease severity is not altered relative to WT controls in mice harboring a gain-of-function mutation in α9 subunits. These findings minimize the likelihood that additional deletion of nAChR α10 subunits impacts disease differently than α9 KO alone, whether through ionotropic, metabotropic, or alternative mechanisms. Moreover, our results provide further evidence of disease-exacerbating roles for nAChR containing α9 subunits (α9*-nAChR) in EAE inflammatory and autoimmune responses. This supports our hypothesis that α9*-nAChR or their downstream mediators are attractive targets for attenuation of inflammation and autoimmunity. Nicotinic acetylcholine receptors (nAChR) are prototypical members of the cys-loop superfamily of neurotransmitter-gated ion channels ,2,3. TheACh has specialized activity as a neurotransmitter in the nervous system, but it also has actions, even early in development, in non-neuronal tissues and organs, such as the immune system ,6. ImmunMaintenance of health via protection against infectious agents and consequences of cell or tissue damage is dependent on normally functioning inflammatory and immune responses ,19,20,21Prompted by our earlier findings that nicotine has immunosuppressive effects, mediated in part by inhibition of T cell differentiation and development , we hypo2+ entry, which in turn could coordinate with, for example, T cell receptor-mediated changes in intracellular Ca2+ signaling to affect cell function . ROS levels also are reduced, by about 30%, in α9/α10 DKO mice (1.5 ± 0.3) relative to WT animals  and defined by Luminol emission levels [illustrated here for animals at disease peak for WT mice, 14 days post-EAE induction; quantified as p/ or homozygous  α9 KI mice. This indicated that the much lower disease severity in the FVB α9 GOF/KI mice cohorts underscores the strong influence of genetic background on biological responses to insults. That aside, across the animals on the FVB background, there was no statistically-significant difference in peak disease severity between WT littermates or α9 GOF/KI hetero- or homo-zygotes  and peak disease at day 15–16, all followed by recovery. Results were informative, in that FVB WT control littermates also had markedly reduced EAE average peak disease severity relative to C57BL/6J WT controls (0.8 ± 0.1 for FVB WT versus 2.4 ± 0.3 for C57BL/6J WT; -zygotes ; Table 2nAChR expressed in the immune and nervous systems afford many mechanisms for modulation of neuroimmune processes ,17,31,32The current study demonstrates that there is attenuation of disease severity and delayed onset of disease symptoms in nAChR α9/α10 DKO mice, as was seen previously in α9 KO or in nicotine-treated mice ,17,31,32Nevertheless, levels of disease attenuation are similar across or within studies for nicotine-treated ,31, α9 KProtection, albeit incomplete, also is seen in nAChR α9/α10 DKO animals against inflammatory cell infiltration into the brains and spinal cords of afflicted animals, and against ROS production in the brain. These effects reported here are observed when peak disease occurs in WT animals. The magnitude of the reduction in inflammatory cell infiltration at that time is slightly lower than the reduction observed in the spinal cords of nicotine-treated animals  assessedCuriously, there is less myelin loss in the brains of WT or in α9/α10 DKO mice than in their spinal cords when measured 30 days after EAE induction and ~2 weeks after peak disease. Moreover, even though there is protection against earlier inflammation and ROS production in DKO animals, myelin loss in the brain is not diminished in DKO mice when measured another ~2 weeks later. This contrasts with the ~50% protection in α9/α10 DKO animals against the more elevated loss of myelin in the spinal cord as seen in this study. Nevertheless, this is comparable to the ~65% reduction in myelin loss in spinal cords from nicotine-treated animals at about the same time in the disease course . Spinal There is very good agreement between our studies done using WT compared to α9 KO, nicotine-treated or α9/α10 DKO mice at different times and with different animal and reagent stocks ,17,31,32+ Tyrc-chPde6b/AntJ background. This underscores the admonition that mouse genetic background matters in terms of responses to treatments and disease susceptibility. It may be of interest to determine whether inflammatory disease resistance is a feature of the FBV strain, whether introduction into those mice of the Pde6b allele to counter development of retinal degeneration and blindness in FBV mice also influences inflammatory and immune response in them, and whether that also is tied to cochlear degeneration and hearing loss.One way in which pro-inflammatory and autoimmune responses mediated by α9*-nAChR could be exacerbated is by elevated expression of heteromeric α9α10-nAChR relative to homomeric α9-nAChR with lower ionotropic function. However, close similarity between effects in the EAE model of α9 KO and of α9/α10 DKO would seem to dismiss this possibility, and although it is yet to be confirmed, peripheral immune cells involved are likely to naturally express the heteromer predominantly, assuming that they also express α10 subunits. Another possible means to elevate α9*-nAChR function, and perhaps to exacerbate EAE, is by introducing a GOF mutation into mice via a KI strategy. This is not literally an approach to provide an inverse of the DKO strategy, although it might be expected that EAE disease could be made worse by hyperactive α9*-nAChR responding to natural cholinergic signals. However, our studies do not show statistically-significant differences in EAE severity and rate or time of onset between WT littermates or nAChR α9 subunit GOF KI heterozygotic or homozygotic mice. The trend toward a slower recovery from peak disease in homozygotic or heterozygotic α9 KI mice might warrant future investigation, but we had expected that if there would be a GOF effect, it would be during the initiation and rise phases of the disease course, when the inflammatory response is most strongly manifest, rather than in the recovery phase, when inflammation is thought to dissipate. Nevertheless, and although the overall level of disease was quite low in our studies, we certainly do not see halving or doubling of effects of α9 KI as we do for α9 KO compared to WT mice. That notwithstanding, it is remarkable that disease severity is less than half of that in WT C57BL/6J mice than in WT animals on the FVB.129P2-Our earlier work suggested that nicotine exposure produces anergy in murine T cells emerging from interactions of hemopoietic stem cells with thymic cells in organ culture ,16. FuncInterestingly, there is no evidence, admittedly in an unnatural vivarium setting, of immunodeficiency or of susceptibility to spontaneous infections in α9 KO or α9/α10 DKO mice. This indicates that there is not wanton destruction of the immune system in these animals, and it is consistent with attenuation, but not total elimination, of disease in the EAE model applied to α9 KO or α9/α10 DKO mice. Similarly, our studies using α9 KI mice are not consistent with animals displaying a hyperinflammatory phenotype, as they show no evidence for hyperimmunity or heightened infection susceptibility. Nevertheless, just as nicotine normalizes mood in a state-dependent manner, blunting depression but also blunting anxiety , nicotinOur findings are consistent with a variety of previous reports showing that exposure to nicotine dampens inflammatory responses in vivo ,57,58. OIndications that nicotine can inhibit inflammation and be immunosuppressive are still relatively new, as are concepts about the so-called “cholinergic anti-inflammatory system” ,65,66,67


The Healthy Brain Rehabilitation Manual’, for patients with a first transient ischaemic attack (TIA) or ‘minor’ stroke of atherosclerotic origin, using the core components of home-based cardiac rehabilitation (CR) and conducted a pilot randomised controlled trial (RCT) to evaluate its effectiveness.I developed a novel home-based rehabilitation programme, including ‘CR is an effective form of secondary prevention for cardiovascular disease. CR after myocardial infarction results in reduced reinfarction risk and all-cause mortality.I followed the Medical Research Council guidelines for developing complex health service interventions. First, I conducted a systematic review (SR) of secondary prevention lifestyle interventions initiated within 90 days of a TIA or ‘minor’ stroke and then a SR on the use of behaviour change techniques (BCTs) in home-based CR. I used the SRs’ findings to adapt a home-based CR manual and design an intervention that was refined following stakeholder input . Then, to assess the applicability of the intervention, I conducted a feasibility study. Patients, recruited from hospital clinics within 4 weeks of a first TIA or minor stroke, were randomly allocated to three groupsFollowing the feasibility study and further intervention refinement, I conducted a 12-week pilot study to test the study protocol before a definitive RCT. Participants, recruited from four different centres, <4 weeks after their first TIA or ‘minor’ stroke, were randomly allocated to: (1) standard care (n=12); (2) CR manual, pedometer and general practitioner follow-up (n=14); and (3) CR manual, pedometer and stroke nurse follow-up (n=14). Follow-up was by telephone at 1, 4 and 9 weeks. Outcome measures were assessed after 12 weeks. Participants’ views on the intervention and research methods were explored using content analysis of poststudy focus group and interview data.My first SR2max testing, which all declined. The intervention was welcomed, and pedometers were valued highly, particularly for goal setting.In the feasibility study,In the pilot study, 35.2% of eligible patients (44/125) consented to contact from a researcher; 90.9% of these (40/44) participated and 97.5% (39/40) completed the study. At 12-week review, cardiovascular risk factors in both intervention arms had improved. Qualitative data confirmed the feasibility and acceptability of the research methods and intervention.The Healthy Brain Rehabilitation Manual’,The study’s recruitment and retention rates, and the intervention’s acceptability and potential effects, indicate that an RCT of a novel home-based CR programme based on ‘


To prove the potential applicability of the fluid in in vitro bioaccessibility test, we compared bioaccessibility of particulates-bound trace elements using this fluid with those of stimulated epithelial lung fluid. Bioaccessibility were relatively low values (<30%) in locus bean substituted lung fluid and stimulated epithelial lung fluid. Specifically, As and Cd had significantly higher bioaccessibility values in locus bean substituted lung fluid than stimulated epithelial lung fluid. The data demonstrate that fluid formulated and used in this study can provide a suitable means of evaluate bioaccessibility of trace elements-bound to airborne particulates.This study was piloted to evaluate bioaccessibility of particulate-bound trace elements using synthetic epithelia lung fluid; in which dipalmitoylphophatidylcholine was substituted with locus bean gum (LBSFL). The resulting data reveal that no significant change in physicochemical characteristics of the stimulated lung fluid compare with similar synthetic fluids; pH value of 7.3, density (0.998gcm•The fluid was used for assessing bioaccessibility of particulate matters-bound trace elements•The formulated fluid can be applied to study in toxicity assessment•The data can be used for inter-laboratory comparison of bioaccessibility of particulate -bound trace element and could stimulate environmental concerns on the impacts of airborne particulates. Specifications TableIn risk assessment, one of the challenges for environmental toxicologist has been development of fluid with properties similar to human tracheobronchial fluids , so as t2SO4), sodium chloride (NaCl), CaCl2, NaHPO4, NaHCO3, KCl, MgCl2, were purchased from Merck, London. Potassium Chloride (KCl), were obtained from BDH. Ascorbic acid, uric acid, glutathione, albumin, cysteine, glycine and polyethylene oxide resin were products of Sigma-Aldrich. All experimental solutions were prepared using double deionized water .All chemicals used in this work were of analytical grade and were used without further purification. Glycine and cysteine were purchased from Sigma Aldrich, UK. Anhydrous sodium sulphates (NaInstruments used in this study included the Inductively Coupled Plasma – Mass Spectrometry (ICPMS), Sciex Elan DRC II, which was operated in standard mode equipped with a Meinhard Concentric Quartz Nebuliser, cyclonic spray chamber, and platinum skimmer and sampler cones.•1000 ml of the fluid was prepared by separate addition of the inorganic phase to organic phase.•2, 150 mg NaHPO4, 2700 mg NaHCO3, 298 mg KCl, 200 mg MgCl2, 72 mg Na2SO4 into a 500 mL HDPE and made up to the volume with ultra-pure water, and then thoroughly mixed.The inorganic phase was prepared by accurately weighed: 6020 mg NaCl, 256 mg CaCl•Similarly, the organic phase was prepared by accurately weighed: 18 mg ascorbic acid, 16 mg uric acid and 30 mg glutathione into a 500 mL HDPE and made up to the volume with ultra-pure water, and then thoroughly mixed.•The two (inorganic and organic) phases were poured into 2 L conical flask containing 260 mg albumin, 122 mg cysteine, 100 mg locus bean gum, 376 mg glycine and 500 mg mucin.•The contents were thoroughly mixed to dissolve all the components.•The physical characteristics of the fluid; pH meter , density (Pycnometer) , conductIn this study, the recipe and procedures for preparation of locus bean substituted fluid was based on those of previously formulated stimulated epithelial lung fluids described in . Table 1in vitro bioaccessibility extraction of particulates-bound trace elements. For this purpose, dataset of total suspended particulate matters collected from different locations at Ado-Ekiti (The formulated LBSLF was used in do-Ekiti  were use0C (1-h shaking followed by 1 h still). The obtained extracts were cooled to room temperature, then centrifuged for 20 min at 3500 rpm and separated for element determination. The percentage bio accessible fraction was calculated as follows [For bioaccessibility study, one quarter of the filter membranes used in samples collection was placed into three different 15 ml polypropylene centrifuge tubes, containing 10 ml each of formulated LBSLF, stimulated epithelia lung fluid (SELF) and artificial lysosome fluid (ALF). The tubes were made airtight and immersed for 2 h in a shaking water bath at 37 follows :(1)PerceThe elemental content of the particulates were determined by Inductively Coupled plasma mass spectrometer (Aligent 5973 inert) (ICPMS). The operating conditions are: plasma flow (18.0 L/min), auxiliary flow (1.80 L/min), Nebulizer flow (1.07 L/min), sheath depth (0.30 L/min), sampling depth (5.00 min), stabilization delay (10 s), power (1.40 kW), pump rate (5 rpm).For quality control, a standard reference material, NIST SRA 1649(Urban particulate matter) was analyzed along with blank filter. The detection limits for the methods were calculated by multiplying the standard deviation of the blank results by 3; in cases where more than one blank was used for an extraction. The concentrations of elements were calculated after subtraction of the blank filter concentration from total concentration in the particulate matters. The recovery of elements was determined by the addition of a spiking solution (prepared using deionized water and standard solution) containing 0.5 mg/L of As, Cd, Cu, Mn, Ni, Zn, Pb were conducted on LBSLF with and without sample. The percentage recovery levels were within 100 ± 15% in all the steps.−3) was almost close to 0.998gcm−3 previous formulation. The conductivity of 13.9 mS m-1 is higher than the reference solution (water) but comparable with 14.5 ± 0.1 mS m-1 previously reported [-1, is slightly lower than 55 mN m-1 reported in earlier study and 72 mN m-1 of reference liquid (water). The use of locust bean gum serves to improve the surface tension of formulated fluid in the present work. The observed high conductivity of the fluid compared to water has been attributed to tonicity effects of the dissolved inorganic salts [The importance of fluids composition on bioaccessibility of different elements has been stretched in several reports . The usereported . Viscosiic salts . Althoug2.5 was considered in this study, as this is the size that can penetrate deeply to the respiratory airways. The bioaccessibility of elements as percentage of element extractable with the formulated fluid in various sites was determined in this study. Only PM