Deciphering salt stress responses in Solanum pimpinellifolium through high-throughput phenotyping.

Soil salinity is a major environmental stressor affecting agricultural productivity worldwide. Understanding plant responses to salt stress is crucial for developing resilient crop varieties. Wild relatives of cultivated crops, such as wild tomato, Solanum pimpinellifolium, can serve as a useful resource to further expand the resilience potential of the cultivated germplasm, S. lycopersicum. In this study, we employed high-throughput phenotyping in the greenhouse and field conditions to explore salt stress responses of a S. pimpinellifolium diversity panel. Our study revealed extensive phenotypic variations in response to salt stress, with traits such as transpiration rate, shoot mass, and ion accumulation showing significant correlations with plant performance. We found that while transpiration was a key determinant of plant performance in the greenhouse, shoot mass strongly correlated with yield under field conditions. Conversely, ion accumulation was the least influential factor under greenhouse conditions. Through a Genome Wide Association Study, we identified candidate genes not previously associated with salt stress, highlighting the power of high-throughput phenotyping in uncovering novel aspects of plant stress responses. This study contributes to our understanding of salt stress tolerance in S. pimpinellifolium and lays the groundwork for further investigations into the genetic basis of these traits, ultimately informing breeding efforts for salinity tolerance in tomato and other crops.

Dietary patterns during pregnancy and maternal and birth outcomes in women with type 1 diabetes: the Environmental Determinants of Islet Autoimmunity (ENDIA) study.

AIMS/HYPOTHESIS: Dietary patterns characterised by high intakes of vegetables may lower the risk of pre-eclampsia and premature birth in the general population. The effect of dietary patterns in women with type 1 diabetes, who have an increased risk of complications in pregnancy, is not known. The aim of this study was to investigate the relationship between dietary patterns and physical activity during pregnancy and maternal complications and birth outcomes in women with type 1 diabetes. We also compared dietary patterns in women with and without type 1 diabetes. METHODS: Diet was assessed in the third trimester using a validated food frequency questionnaire in participants followed prospectively in the multi-centre Environmental Determinants of Islet Autoimmunity (ENDIA) study. Dietary patterns were characterised by principal component analysis. The Pregnancy Physical Activity Questionnaire was completed in each trimester. Data for maternal and birth outcomes were collected prospectively. RESULTS: Questionnaires were completed by 973 participants during 1124 pregnancies. Women with type 1 diabetes (n=615 pregnancies with dietary data) were more likely to have a 'fresh food' dietary pattern than women without type 1 diabetes (OR 1.19, 95% CI 1.07, 1.31; p=0.001). In women with type 1 diabetes, an increase equivalent to a change from quartile 1 to 3 in 'fresh food' dietary pattern score was associated with a lower risk of pre-eclampsia (OR 0.37, 95% CI 0.17, 0.78; p=0.01) and premature birth (OR 0.35, 95% CI 0.20, 0.62, p<0.001). These associations were mediated in part by BMI and HbA1c. The 'processed food' dietary pattern was associated with an increased birthweight (ß coefficient 56.8 g, 95% CI 2.8, 110.8; p=0.04). Physical activity did not relate to outcomes. CONCLUSIONS/INTERPRETATION: A dietary pattern higher in fresh foods during pregnancy was associated with sizeable reductions in risk of pre-eclampsia and premature birth in women with type 1 diabetes.

Genetic mapping of the early responses to salt stress in Arabidopsis thaliana.

Salt stress decreases plant growth prior to significant ion accumulation in the shoot. However, the processes underlying this rapid reduction in growth are still unknown. To understand the changes in salt stress responses through time and at multiple physiological levels, examining different plant processes within a single set-up is required. Recent advances in phenotyping has allowed the image-based estimation of plant growth, morphology, colour and photosynthetic activity. In this study, we examined the salt stress-induced responses of 191 Arabidopsis accessions from 1 h to 7 days after treatment using high-throughput phenotyping. Multivariate analyses and machine learning algorithms identified that quantum yield measured in the light-adapted state (Fv' /Fm' ) greatly affected growth maintenance in the early phase of salt stress, whereas the maximum quantum yield (QYmax ) was crucial at a later stage. In addition, our genome-wide association study (GWAS) identified 770 loci that were specific to salt stress, in which two loci associated with QYmax and Fv' /Fm' were selected for validation using T-DNA insertion lines. We characterized an unknown protein kinase found in the QYmax locus that reduced photosynthetic efficiency and growth maintenance under salt stress. Understanding the molecular context of the candidate genes identified will provide valuable insights into the early plant responses to salt stress. Furthermore, our work incorporates high-throughput phenotyping, multivariate analyses and GWAS, uncovering details of temporal stress responses and identifying associations across different traits and time points, which are likely to constitute the genetic components of salinity tolerance.

Decreased occurrence of ketoacidosis and preservation of beta cell function in relatives screened and monitored for type 1 diabetes in Australia and New Zealand.

AIMS: Islet autoantibody screening of infants and young children in the Northern Hemisphere, together with semi-annual metabolic monitoring, is associated with a lower risk of ketoacidosis (DKA) and improved glucose control after diagnosis of clinical (stage 3) type 1 diabetes (T1D). We aimed to determine if similar benefits applied to older Australians and New Zealanders monitored less rigorously. METHODS: DKA occurrence and metabolic control were compared between T1D relatives screened and monitored for T1D and unscreened individuals diagnosed in the general population, ascertained from the Australasian Diabetes Data Network. RESULTS: Between 2005 and 2019, 17,105 relatives (mean (SD) age 15.7 (10.8) years; 52% female) were screened for autoantibodies against insulin, glutamic acid decarboxylase, and insulinoma-associated protein 2. Of these, 652 screened positive to a single and 306 to multiple autoantibody specificities, of whom 201 and 215, respectively, underwent metabolic monitoring. Of 178 relatives diagnosed with stage 3 T1D, 9 (5%) had DKA, 7 of whom had not undertaken metabolic monitoring. The frequency of DKA in the general population was 31%. After correction for age, sex and T1D family history, the frequency of DKA in screened relatives was >80% lower than in the general population. HbA1c and insulin requirements following diagnosis were also lower in screened relatives, consistent with greater beta cell reserve. CONCLUSIONS: T1D autoantibody screening and metabolic monitoring of older children and young adults in Australia and New Zealand, by enabling pre-clinical diagnosis when beta cell reserve is greater, confers protection from DKA. These clinical benefits support ongoing efforts to increase screening activity in the region and should facilitate the application of emerging immunotherapies.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

BACKGROUND: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. METHODS: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. RESULTS: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. CONCLUSION: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

High-throughput 3D modelling to dissect the genetic control of leaf elongation in barley (Hordeum vulgare).

To optimize shoot growth and structure of cereals, we need to understand the genetic components controlling initiation and elongation. While measuring total shoot growth at high throughput using 2D imaging has progressed, recovering the 3D shoot structure of small grain cereals at a large scale is still challenging. Here, we present a method for measuring defined individual leaves of cereals, such as wheat and barley, using few images. Plant shoot modelling over time was used to measure the initiation and elongation of leaves in a bi-parental barley mapping population under low and high soil salinity. We detected quantitative trait loci (QTL) related to shoot growth per se, using both simple 2D total shoot measurements and our approach of measuring individual leaves. In addition, we detected QTL specific to leaf elongation and not to total shoot size. Of particular importance was the detection of a QTL on chromosome 3H specific to the early responses of leaf elongation to salt stress, a locus that could not be detected without the computer vision tools developed in this study.

Stimulation of homologous recombination in plants expressing heterologous recombinases.

BACKGROUND: Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants. RESULTS: We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive ß-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility. CONCLUSION: Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.

Changes in pancreatic exocrine function in young at-risk children followed to islet autoimmunity and type 1 diabetes in the ENDIA study.

BACKGROUNDS: We aimed to monitor pancreatic exocrine function longitudinally in relation to the development of islet autoimmunity (IA) and type 1 diabetes (T1D) in at-risk children with a first-degree relative with T1D, who were followed prospectively in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. METHODS: Fecal elastase-1 (FE-1) concentration was measured longitudinally in 85 ENDIA children from median age 1.0 (IQR 0.7,1.3) year. Twenty-eight of 85 children (progressors) developed persistent islet autoantibodies at median age of 1.5 (IQR 1.1,2.5) years, of whom 11 went on to develop clinical diabetes. The other 57 islet autoantibody-negative children (non-progressors) followed similarly were age and gender-matched with the progressors. An adjusted linear mixed model compared FE-1 concentrations in progressors and non-progressors. RESULTS: Baseline FE-1 did not differ between progressors and non-progressors, or by HLA DR type or proband status. FE-1 decreased over time in progressors in comparison to non-progressors (Wald statistic 5.46, P = .02); in some progressors the fall in FE-1 preceded the onset of IA. CONCLUSIONS: Pancreatic exocrine function decreases in the majority of young at-risk children who progress to IA and T1D.

RNAi-suppression of barley caffeic acid O-methyltransferase modifies lignin despite redundancy in the gene family.

Caffeic acid O-methyltransferase (COMT), the lignin biosynthesis gene modified in many brown-midrib high-digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl-to-guaiacyl ratio was reduced by ~50%, the 5-hydroxyguaiacyl (5-OH-G) unit incorporated into lignin at 10--15-fold higher levels than normal, and the amount of p-coumaric acid ester-linked to cell walls was reduced by ~50%. No brown-midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown-midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.

The synaptonemal complex protein ZYP1 is required for imposition of meiotic crossovers in barley.

In many cereal crops, meiotic crossovers predominantly occur toward the ends of chromosomes and 30 to 50% of genes rarely recombine. This limits the exploitation of genetic variation by plant breeding. Previous reports demonstrate that chiasma frequency can be manipulated in plants by depletion of the synaptonemal complex protein ZIPPER1 (ZYP1) but conflict as to the direction of change, with fewer chiasmata reported in Arabidopsis thaliana and more crossovers reported for rice (Oryza sativa). Here, we use RNA interference (RNAi) to reduce the amount of ZYP1 in barley (Hordeum vulgare) to only 2 to 17% of normal zygotene levels. In the ZYP1(RNAi) lines, fewer than half of the chromosome pairs formed bivalents at metaphase and many univalents were observed, leading to chromosome nondisjunction and semisterility. The number of chiasmata per cell was reduced from 14 in control plants to three to four in the ZYP1-depleted lines, although the localization of residual chiasmata was not affected. DNA double-strand break formation appeared normal, but the recombination pathway was defective at later stages. A meiotic time course revealed a 12-h delay in prophase I progression to the first labeled tetrads. Barley ZYP1 appears to function similarly to ZIP1/ZYP1 in yeast and Arabidopsis, with an opposite effect on crossover number to ZEP1 in rice, another member of the Poaceae.

The effect of temperature on the male and female recombination landscape of barley.

Barley (Hordeum vulgare) is a crop of global significance. However, a third of the genes of barley are largely inaccessible to conventional breeding programmes as crossovers are localised to the ends of the chromosomes. This work examines whether crossovers can be shifted to more proximal regions simply by elevating growth temperature. We utilised a genome-wide marker set for linkage analysis combined with cytological mapping of crossover events to examine the recombination landscape of plants grown at different temperatures. We found that barley shows heterochiasmy, that is, differences between female and male recombination frequencies. In addition, we found that elevated temperature significantly changes patterns of recombination in male meiosis only, with a repositioning of Class I crossovers determined by cytological mapping of HvMLH3 foci. We show that the length of synaptonemal complexes in male meiocytes increases in response to temperature. The results demonstrate that the distribution of crossover events are malleable and can be shifted to proximal regions by altering the growth temperature. The shift in recombination is the result of altering the distribution of Class I crossovers, but the higher recombination at elevated temperatures is potentially not the result of an increase in Class I events.

A genome wide association scan for (1,3;1,4)-ß-glucan content in the grain of contemporary 2-row Spring and Winter barleys.

BACKGROUND: (1,3;1,4)-ß-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-ß-glucan are required for brewing and distilling and high levels for positive impacts on human health. RESULTS: We quantified grain (1,3;1,4)-ß-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes. CONCLUSIONS: We show that a region of the genome on chromosome 2 containing a cluster of CELLULOSE SYNTHASE-LIKE (Csl) genes, including CslF3, CslF4, CslF8, CslF10, CslF12 and CslH, as well as a region on chromosome 1H containing CslF9, are associated with the phenotype in this germplasm. We also observed that several regions identified by GWAS contain glycoside hydrolases that are possibly involved in (1,3;1,4)-ß-glucan breakdown, together with other genes that might participate in (1,3;1,4)-ß-glucan synthesis, re-modelling or regulation. This analysis provides new opportunities for understanding the genes related to the regulation of (1,3;1,4)-ß-glucan content in cereal grains.

Genetic parameters for yearling male reproduction traits in tropical composite cattle population.

Fertility is economically important but is hard to quantify and measure in breeding programs which has led extensive breeding programs to ignore fertility in their selection criteria. While female fertility traits have been extensively researched, male fertility traits have been largely ignored. It is estimated that 20% to 40% of bulls have sub-fertility, reducing the number of calves born and profits, highlighting the importance of investigating bull fertility. The most practical measure of male fertility is a bull breeding soundness evaluation (BBSE) which assesses structure as well as semen quality and quantity. Generally, traits recorded in a BBSE are neither genetically evaluated nor used for selection in breeding programs. All traits recorded during a BBSE were analyzed through a series of univariate and bivariate linear mixed models using a genomic relationship matrix to estimate genetic parameters. All genotype and phenotype data were obtained from a tropical composite commercial cattle population and imputed to 27,638 single-nucleotide polymorphisms (SNPs) with a total of 2,613 genotyped animals with BBSE records ranging from 616 to 826 animals depending on the trait. The heritabilities of the 27 traits recorded during a BBSE ranged from 0.02 to 0.49. Seven of the male fertility traits were recommended to be included in a breeding program based on their heritability and their phenotypic and genetic correlations. These traits are scrotal circumference, percent normal sperm, proximal droplets, distal midpiece reflex, knobbed acrosomes, vacuoles/teratoids, and sheath score. Using these seven traits in a breeding program would result in higher calving rates, increasing production and profitability.

One of the key profit drivers in any animal breeding program is fertility as it contributes directly to the progeny produced. Typically, fertility traits are hard to quantify and lowly heritable so they are often ignored in breeding programs. The inclusion of male fertility traits could allow for selection on heritable traits that are easy to measure and implement in a commercial breeding program. The utilization of male fertility traits could improve overall fertility and production. Bull breeding soundness evaluation traits were heritable, ranging from low to high, allowing for genetic improvement in those traits. Seven traits were recommended as selection criteria in a breeding program, which included two physical traits and five sperm traits. Implementing these seven traits in a breeding program would allow for a higher calving rate and associated increased profits.

Early Dysglycemia Is Detectable Using Continuous Glucose Monitoring in Very Young Children at Risk of Type 1 Diabetes.

OBJECTIVE: Continuous glucose monitoring (CGM) can detect early dysglycemia in older children and adults with presymptomatic type 1 diabetes (T1D) and predict risk of progression to clinical onset. However, CGM data for very young children at greatest risk of disease progression are lacking. This study aimed to investigate the use of CGM data measured in children being longitudinally observed in the Australian Environmental Determinants of Islet Autoimmunity (ENDIA) study from birth to age 10 years. RESEARCH DESIGN AND METHODS: Between January 2021 and June 2023, 31 ENDIA children with persistent multiple islet autoimmunity (PM Ab+) and 24 age-matched control children underwent CGM assessment alongside standard clinical monitoring. The CGM metrics of glucose SD (SDSGL), coefficient of variation (CEV), mean sensor glucose (SGL), and percentage of time >7.8 mmol/L (>140 mg/dL) were determined and examined for between-group differences. RESULTS: The mean (SD) ages of PM Ab+ and Ab- children were 4.4 (1.8) and 4.7 (1.9) years, respectively. Eighty-six percent of eligible PM Ab+ children consented to CGM wear, achieving a median (quartile 1 [Q1], Q3) sensor wear period of 12.5 (9.0, 15.0) days. PM Ab+ children had higher median (Q1, Q3) SDSGL (1.1 [0.9, 1.3] vs. 0.9 [0.8, 1.0] mmol/L; P < 0.001) and CEV (17.3% [16.0, 20.9] vs. 14.7% [12.9, 16.6]; P < 0.001). Percentage of time >7.8 mmol/L was greater in PM Ab+ children (median [Q1, Q3] 8.0% [4.4, 13.0] compared with 3.3% [1.4, 5.3] in Ab- children; P = 0.005). Mean SGL did not differ significantly between groups (P = 0.10). CONCLUSIONS: CGM is feasible and well tolerated in very young children at risk of T1D. Very young PM Ab+ children have increased SDSGL, CEV, and percentage of time >7.8 mmol/L, consistent with prior studies involving older participants.

Environmental Determinants of Islet Autoimmunity (ENDIA) longitudinal prospective pregnancy to childhood cohort study of Australian children at risk of type 1 diabetes: parental demographics and birth information.

INTRODUCTION: The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is an ongoing Australian prospective cohort study investigating how modifiable prenatal and early-life exposures drive the development of islet autoimmunity and type 1 diabetes (T1D) in children. In this profile, we describe the cohort's parental demographics, maternal and neonatal outcomes and human leukocyte antigen (HLA) genotypes. RESEARCH DESIGN AND METHODS: Inclusion criteria were an unborn child, or infant aged less than 6 months, with a first-degree relative (FDR) with T1D. The primary outcome was persistent islet autoimmunity, with children followed until a T1D diagnosis or 10 years of age. Demographic data were collected at enrollment. Lifestyle, clinical and anthropometric data were collected at each visit during pregnancy and clinical pregnancy and birth data were verified against medical case notes. Data were compared between mothers with and without T1D. HLA genotyping was performed on the ENDIA child and all available FDRs. RESULTS: The final cohort comprised 1473 infants born to 1214 gestational mothers across 1453 pregnancies, with 80% enrolled during pregnancy. The distribution of familial T1D probands was 62% maternal, 28% paternal and 11% sibling. The frequency of high-risk HLA genotypes was highest in T1D probands, followed by ENDIA infants, and lowest among unaffected family members. Mothers with T1D had higher rates of pregnancy complications and perinatal intervention, and larger babies of shorter gestation. Parent demographics were comparable to the Australian population for age, parity and obesity. A greater percentage of ENDIA parents were Australian born, lived in a major city and had higher socioeconomic advantage and education. CONCLUSIONS: This comprehensive profile provides the context for understanding ENDIA's scope, methodology, unique strengths and limitations. Now fully recruited, ENDIA will provide unique insights into the roles of early-life factors in the development of islet autoimmunity and T1D in the Australian environment. TRIAL REGISTRATION NUMBER: ACTRN12613000794707.

A Long-Term Evaluation of Facebook for Recruitment and Retention in the ENDIA Type 1 Diabetes Pregnancy-Birth Cohort Study.

BACKGROUND: The Environmental Determinants of Islet Autoimmunity (ENDIA) study is an Australia-wide pregnancy-birth cohort study following children who have a first-degree relative with type 1 diabetes (ACTRN1261300794707). A dedicated ENDIA Facebook page was established in 2013 with the aim of enhancing recruitment and supporting participant retention through dissemination of study information. To measure the impact of Facebook, we evaluated the sources of referral to the study, cohort demographics, and withdrawal rates. We also investigated whether engagement with Facebook content was associated with specific post themes. METHODS: Characteristics of Facebook versus conventional recruits were compared using linear, logistic, and multinomial logistic regression models. Logistic regression was used to determine the risk of study withdrawal. Data pertaining to 794 Facebook posts over 7.5 years were included in the analysis. RESULTS: Facebook was the third largest source of referral (300/1511; 19.9%). Facebook recruits were more frequently Australian-born (P < .001) enrolling postnatally (P = .01) and withdrew from the study at a significantly lower rate compared with conventional recruits (4.7% vs 12.3%; P < .001) after a median of follow-up of 3.3 years. Facebook content featuring stories and images of participants received the highest engagement even though <20% of the 2337 Facebook followers were enrolled in the study. CONCLUSIONS: Facebook was a valuable recruitment tool for ENDIA. Compared with conventional recruits, Facebook recruits were three times less likely to withdraw during long-term follow-up and had different sociodemographic characteristics. Facebook content featuring participants was the most engaging. These findings inform social media strategies for future cohort and type 1 diabetes studies. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry: ACTRN1261300794707.

Experiences of Caregivers and At-Risk Children Enrolled in a Prospective Pregnancy-Birth Cohort Study into the Causes of Type 1 Diabetes: The ENDIA Study.

BACKGROUND: We sought research experiences of caregivers and their children were enrolled in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. METHODS: ENDIA is a pregnancy-birth cohort investigating early-life causes of type 1 diabetes (T1D). Surveys were sent to 1090 families between June 2021 and March 2022 with a median participation of >5 years. Caregivers completed a 12-item survey. Children ≥ 3 years completed a four-item survey. RESULTS: The surveys were completed by 550/1090 families (50.5%) and 324/847 children (38.3%). The research experience was rated as either "excellent" or "good" by 95% of caregivers, and 81% of children were either "ok", "happy" or "very happy". The caregivers were motivated by contributing to research and monitoring their children for T1D. Relationships with the research staff influenced the experience. The children most liked virtual reality headsets, toys, and "helping". Blood tests were least liked by the children and were the foremost reason that 23.4% of the caregivers considered withdrawing. The children valued gifts more than their caregivers. Only 5.9% of responses indicated dissatisfaction with some aspects of the protocol. The self-collection of samples in regional areas, or during the COVID-19 pandemic restrictions, were accepted. CONCLUSIONS: This evaluation identified modifiable protocol elements and was conducted to further improve satisfaction. What was important to the children was distinct from their caregivers.

A surge in serum mucosal cytokines associated with seroconversion in children at risk for type 1 diabetes.

AIMS/INTRODUCTION: Autoantibodies to pancreatic islet antigens identify young children at high risk of type 1 diabetes. On a background of genetic susceptibility, islet autoimmunity is thought to be driven by environmental factors, of which enteric viruses are prime candidates. We sought evidence for enteric pathology in children genetically at-risk for type 1 diabetes followed from birth who had developed islet autoantibodies ("seroconverted"), by measuring mucosa-associated cytokines in their sera. MATERIALS AND METHODS: Sera were collected 3 monthly from birth from children with a first-degree type 1 diabetes relative, in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Children who seroconverted were matched for sex, age, and sample availability with seronegative children. Luminex xMap technology was used to measure serum cytokines. RESULTS: Of eight children who seroconverted, for whom serum samples were available at least 6 months before and after seroconversion, the serum concentrations of mucosa-associated cytokines IL-21, IL-22, IL-25, and IL-10, the Th17-related cytokines IL-17F and IL-23, as well as IL-33, IFN-γ, and IL-4, peaked from a low baseline in seven around the time of seroconversion and in one preceding seroconversion. These changes were not detected in eight sex- and age-matched seronegative controls, or in a separate cohort of 11 unmatched seronegative children. CONCLUSIONS: In a cohort of children at risk for type 1 diabetes followed from birth, a transient, systemic increase in mucosa-associated cytokines around the time of seroconversion lends support to the view that mucosal infection, e.g., by an enteric virus, may drive the development of islet autoimmunity.

Protocol for a nested case-control study design for omics investigations in the Environmental Determinants of Islet Autoimmunity cohort.

Background: The Environmental Determinants of Islet Autoimmunity (ENDIA) pregnancy-birth cohort investigates the developmental origins of type 1 diabetes (T1D), with recruitment between 2013 and 2019. ENDIA is the first study in the world with comprehensive data and biospecimen collection during pregnancy, at birth and through childhood from at-risk children who have a first-degree relative with T1D. Environmental exposures are thought to drive the progression to clinical T1D, with pancreatic islet autoimmunity (IA) developing in genetically susceptible individuals. The exposures and key molecular mechanisms driving this progression are unknown. Persistent IA is the primary outcome of ENDIA; defined as a positive antibody for at least one of IAA, GAD, ZnT8 or IA2 on two consecutive occasions and signifies high risk of clinical T1D.Method: A nested case-control (NCC) study design with 54 cases and 161 matched controls aims to investigate associations between persistent IA and longitudinal omics exposures in ENDIA. The NCC study will analyse samples obtained from ENDIA children who have either developed persistent IA or progressed to clinical T1D (cases) and matched control children at risk of developing persistent IA. Control children were matched on sex and age, with all four autoantibodies absent within a defined window of the case's onset date. Cases seroconverted at a median of 1.37 years (IQR 0.95, 2.56). Longitudinal omics data generated from approximately 16,000 samples of different biospecimen types, will enable evaluation of changes from pregnancy through childhood.Conclusions: This paper describes the ENDIA NCC study, omics platform design considerations and planned univariate and multivariate analyses for its longitudinal data. Methodologies for multivariate omics analysis with longitudinal data are discovery-focused and data driven. There is currently no single multivariate method tailored specifically for the longitudinal omics data that the ENDIA NCC study will generate and therefore omics analysis results will require either cross validation or independent validation.KEY MESSAGESThe ENDIA nested case-control study will utilize longitudinal omics data on approximately 16,000 samples from 190 unique children at risk of type 1 diabetes (T1D), including 54 who have developed islet autoimmunity (IA), followed during pregnancy, at birth and during early childhood, enabling the developmental origins of T1D to be explored.