Intestinal dysbiosis and inflammation in children with repaired esophageal atresia.

OBJECTIVES: This study aims to compare the intestinal microbiota and intestinal inflammation of children with esophageal atresia (EA) to matched healthy controls, and to investigate the relationship between these factors and clinical outcomes. METHODS: A cross-sectional study of 35 children with EA and 35 matched healthy controls (HC) from a single tertiary pediatric hospital in Australia was conducted. Demographic and dietary data were collected using surveys. Stool samples were analyzed using 16S rRNA sequencing, and fecal calprotectin measurements were used to measure intestinal inflammation. Comparisons were made between the groups, and correlations between the microbiota and clinical factors were investigated in the EA cohort. RESULTS: Compared to HC, children with EA had similar alpha diversity, but beta diversity analysis revealed clustering of EA and HC cohorts. Children with EA had a significantly higher relative abundance of the order Lactobacillales, and a lower abundance of the genus uncultured Bacteroidales S24-7. Fecal calprotectin was significantly higher in children with EA compared to HC. In the EA cohort, children taking proton pump inhibitors (PPI's) had lower alpha diversity and higher calprotectin levels compared to those not taking PPI's. There was a negative correlation between calprotectin and length/height-for-age z scores, and children with higher calprotectin levels had a greater burden of gastrointestinal symptoms. CONCLUSIONS: Children with EA have an altered intestinal microbiota compared to HC, which is likely related to PPI use, and may be impacting on growth and quality of life. It is important to rationalize PPI use in this cohort.

Evaluating the Dietary Intake of Children With Esophageal Atresia: A Prospective, Controlled, Observational Study.

OBJECTIVES: Children with esophageal atresia (EA) often have feeding difficulties and dysphagia, which may compromise their nutritional status. This study aimed to compare dietary intake between children with EA and matched healthy controls (HC) and to investigate the relationship between dietary factors, growth, dysphagia, and feeding difficulties in the EA cohort. METHODS: This cross-sectional cohort study recruited children with EA and HC aged 2-17 years from a tertiary pediatric hospital in Australia. Growth parameters were measured. Dietary intake was assessed using the validated Australian Child and Adolescent Eating Survey. Dysphagia and feeding difficulties were assessed using objective questionnaires. RESULTS: Twenty-one children with EA were matched for age and sex with 21 HC. Compared to HC, children with EA had lower mean z scores for height-for-age, but mean weight-for-age and body mass index-for-age z scores were similar. Energy intake was similar between the groups. The diet of children with EA consisted of a higher proportion of fats and lower proportion of carbohydrates compared to matched HC. Dysphagia severity in children with EA positively correlated with proportion of energy from fats and saturated fats. CONCLUSIONS: Children with EA have similar energy intake and growth parameters to HC, but their diet consists of a higher proportion of fats and lower proportion of carbohydrates compared to HC. Targeted dietary interventions and parental education are necessary.

Transcriptional modification of host cells harboring Toxoplasma gondii bradyzoites prevents IFN gamma-mediated cell death.

Toxoplasma gondii develops a latent infection in the muscle and central nervous system that acts as a reservoir for acute-stage reactivation in vulnerable patients. Little is understood about how parasites manipulate host cells during latent infection and the impact this has on survival. We show that bradyzoites impart a unique transcriptional signature on infected host cells. Many of these transcriptional changes rely on protein export and result in the suppression of type I interferon (IFN) and IFNγ signaling more so than in acute stages. Loss of the protein export component, MYR1, abrogates transcriptional remodeling and prevents suppression of IFN signaling. Among the exported proteins, the inhibitor of STAT1 transcription (IST) plays a key role in limiting IFNγ signaling in bradyzoites. Furthermore, bradyzoite protein export protects host cells from IFNγ-mediated cell death, even when export is restricted to latent stages. These findings highlight the functional importance of host manipulation in Toxoplasma's bradyzoite stages.

Paediatric pancreatic diseases.

Paediatric pancreatic diseases are often under-recognised and may be associated with severe diseases and significant clinical consequences. In recent years, advances have been made in key areas, particularly with the contributions from international societies and study groups focused on paediatric pancreatic disease research. This review focuses on the two key manifestations of pancreatic disorders in childhood, pancreatitis and exocrine pancreatic dysfunction.

The intestinal virome in children with cystic fibrosis differs from healthy controls.

Intestinal bacterial dysbiosis is evident in children with cystic fibrosis (CF) and intestinal viruses may be contributory, given their influence on bacterial species diversity and biochemical cycles. We performed a prospective, case-control study on children with CF and age and gender matched healthy controls (HC), to investigate the composition and function of intestinal viral communities. Stool samples were enriched for viral DNA and RNA by viral extraction, random amplification and purification before sequencing (Illumina MiSeq). Taxonomic assignment of viruses was performed using Vipie. Functional annotation was performed using Virsorter. Inflammation was measured by calprotectin and M2-pyruvate kinase (M2-PK). Eight CF and eight HC subjects were included (50% male, mean age 6.9 ± 3.0 and 6.4 ± 5.3 years, respectively, p = 0.8). All CF subjects were pancreatic insufficient. Regarding the intestinal virome, no difference in Shannon index between CF and HC was identified. Taxonomy-based beta-diversity (presence-absence Bray-Curtis dissimilarity) was significantly different between CF and HC (R2 = 0.12, p = 0.001). Myoviridae, Faecalibacterium phage FP Taranis and unclassified Gokushovirinae were significantly decreased in CF compared with HC (q<0.05). In children with CF (compared to HC), the relative abundance of genes annotated to (i) a peptidoglycan-binding domain of the peptidoglycan hydrolases (COG3409) was significantly increased (q<0.05) and (ii) capsid protein (F protein) (PF02305.16) was significantly decreased (q<0.05). Picornavirales, Picornaviridae, and Enterovirus were found to positively correlate with weight and BMI (r = 0.84, q = 0.01). Single-stranded DNA viruses negatively correlated with M2-PK (r = -0.86, q = 0.048). Children with CF have an altered intestinal virome compared to well-matched HC, with both taxonomic and predicted functional changes. Further exploration of Faecalibacterium phages, Gokushovirinae and phage lysins are warranted. Intestinal viruses and their functions may have important clinical implications for intestinal inflammation and growth in children with CF, potentially providing novel therapeutic targets.

Evaluating the Dietary Intakes of Energy, Macronutrients, Sugar, Fiber, and Micronutrients in Children With Celiac Disease.

OBJECTIVES: Children with celiac disease (CD) follow a lifelong gluten-free diet. This restrictive diet may be associated with nutritional compromise. Our objectives were, therefore, to evaluate the dietary composition (energy, macronutrients and micronutrients, and fiber) in children with CD compared with healthy controls (HC) and relationship between dietary composition and socioeconomic status. METHODS: This cross-sectional, case-control study recruited children with CD ages 2 to 18 years and HC matched for age, sex, and socioeconomic status. Clinical, sociodemographic, and dietary information were collected. A false discovery rate correction was applied to the P-value for multiple comparisons (q-value). RESULTS: Sixty-five CD children were matched with 65 HC (mean [SD] age: 10.2 [3.6] vs 10.1 [3.7] years, P = 0.96). Compared with HC, CD children had higher intakes of energy (2413.2 [489.9] vs 2190.8 (593.5) kcal/day, P = 0.02), total fat (818.1 ±â€Š180.9 vs 714.3 ±â€Š212.2 kcal/day, q = 0.018), and subtypes of fat (saturated, polyunsaturated, and monounsaturated). There were no differences in other macronutrients, sugar, micronutrients, or fiber between CD and HC, and no difference in dietary intake among CD between socioeconomic disadvantage versus advantage. Children with CD had lower weight z-scores (-0.06 [1.05] vs 0.47 [0.96], P = 0.003) and body mass index (BMI) z-scores (-0.02 [0.88] vs 0.41 [1.09], P = 0.02) than HC. CONCLUSIONS: Children with CD had higher calorie and fat intake compared with HC. Despite this, CD children had lower weight and BMI z-scores compared with HC.

Cystic Fibrosis-related Liver Disease is Associated With Increased Disease Burden and Endocrine Comorbidities.

BACKGROUND: Cystic fibrosis-related liver disease (CFLD) is the leading nonpulmonary cause of mortality in cystic fibrosis (CF). We evaluated and compared the burden of disease and nonrespiratory comorbidities of those with severe CFLD and those without (noCFLD). METHODS: A retrospective nationwide (Australia) longitudinal review (from 1998 to 2016) of severe CFLD patients compared with noCFLD controls (matched 1 : 1 for age, genotype, pancreatic insufficiency, and center). RESULTS: One hundred sixty-six patients with severe CFLD and 166 with noCFLD were identified. Forced expiratory volume in 1 second percentage of predicted (FEV1%) was significantly lower in CFLD than noCFLD across all ages (estimate [SE] -6.05% [2.12]; P = 0.004). Median (IQR) hospitalizations per patient per year were higher in CFLD than noCFLD for: respiratory indications (0.6 [0.2-1.3] vs 0.4 [0.1-0.9]; P = 0.002); gastrointestinal indications (0.09 [0-0.2] vs 0 [0-0.05]; P < 0.001); and other indications (0.05 [0-0.2] vs 0 [0-0.1]; P = 0.03). In the CFLD cohort, there was increased use of nasogastric (12.6% vs 5.4%; OR 2.51 [95% CI 1.06-6.46]; P = 0.03) and gastrostomy nutritional supplementation (22.9% vs 13.2%; OR 1.93 [95% CI 1.05-3.63]; P = 0.03). Additionally, the CFLD cohort had a higher frequency of bone diseases, osteopenia (26.5% vs 16.8%; OR 1.77 [95%CI 1.01-3.15]; P = 0.04) and osteoporosis (16.2% vs 8.4%; OR 2.1 [95% CI 1.01-4.52]; P = 0.04), as well as CF-related diabetes (38.5% vs 19.2%; OR 2.61 [95% CI 1.55-4.47]; P = 0.001). CONCLUSIONS: Patients with severe CFLD have greater disease burden, with higher number of hospitalizations (both respiratory and nonrespiratory indications), nutritional interventions, and are at higher risk of CF-related bone disease and diabetes.

Evaluating the Alimentary and Respiratory Tracts in Health and disease (EARTH) research programme: a protocol for prospective, longitudinal, controlled, observational studies in children with chronic disease at an Australian tertiary paediatric hospital.

INTRODUCTION: Chronic gastrointestinal and respiratory conditions of childhood can have long-lasting physical, psychosocial and economic effects on children and their families. Alterations in diet and intestinal and respiratory microbiomes may have important implications for physical and psychosocial health. Diet influences the intestinal microbiome and should be considered when exploring disease-specific alterations. The concepts of gut-brain and gut-lung axes provide novel perspectives for examining chronic childhood disease(s). We established the 'Evaluating the Alimentary and Respiratory Tracts in Health and disease' (EARTH) research programme to provide a structured, holistic evaluation of children with chronic gastrointestinal and/or respiratory conditions. METHODS AND ANALYSIS: The EARTH programme provides a framework for a series of prospective, longitudinal, controlled, observational studies (comprised of individual substudies), conducted at an Australian tertiary paediatric hospital (the methodology is applicable to other settings). Children with a chronic gastrointestinal and/or respiratory condition will be compared with age and gender matched healthy controls (HC) across a 12-month period. The following will be collected at baseline, 6 and 12 months: (i) stool, (ii) oropharyngeal swab/sputum, (iii) semi-quantitative food frequency questionnaire, (iv) details of disease symptomatology, (v) health-related quality of life and (vi) psychosocial factors. Data on the intestinal and respiratory microbiomes and diet will be compared between children with a condition and HC. Correlations between dietary intake (energy, macro-nutrients and micro-nutrients), intestinal and respiratory microbiomes within each group will be explored. Data on disease symptomatology, quality of life and psychosocial factors will be compared between condition and HC cohorts.Results will be hypothesis-generating and direct future focussed studies. There is future potential for direct translation into clinical care, as diet is a highly modifiable factor. ETHICS AND DISSEMINATION: Ethics approval: Sydney Children's Hospitals Network Human Research Ethics Committee (HREC/18/SCHN/26). Results will be presented at international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04071314.

Probiotics for people with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a multisystem disease and the importance of growth and nutrition has been well established, given its implications for lung function and overall survival. It has been established that intestinal dysbiosis (i.e. microbial imbalance) and inflammation is present in people with CF. Probiotics are commercially available (over-the-counter) and may improve both intestinal and overall health. OBJECTIVES: To assess the efficacy and safety of probiotics for improving health outcomes in children and adults with CF. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. Date of last register search: 20 January 2020. We also searched ongoing trials registries and the reference lists of relevant articles and reviews. Date of last search: 29 January 2019. SELECTION CRITERIA: Randomised or quasi-randomised controlled trials (RCTs) assessing efficacies and safety of probiotics in children and adults with CF. Cross-over RCTs with a washout phase were included and for those without a washout period, only the first phase of each trial was analysed. DATA COLLECTION AND ANALYSIS: We independently extracted data and assessed the risk of bias of the included trials; we used GRADE to assess the certainty of the evidence. We contacted trial authors for additional data. Meta-analyses were undertaken on outcomes at several time points. MAIN RESULTS: We identified 17 trials and included 12 RCTs (11 completed and one trial protocol - this trial was terminated early) (464 participants). Eight trials included only children, whilst four trials included both children and adults. Trial duration ranged from one to 12 months. Nine trials compared a probiotic (seven single strain and three multistrain preparations) with a placebo preparation, two trials compared a synbiotic (multistrain) with a placebo preparation and one trial compared two probiotic preparations. Overall we judged the risk of bias in the 12 trials to be low. Three trials had a high risk of performance bias, two trials a high risk of attrition bias and six trials a high risk of reporting bias. Only two trials were judged to have low or unclear risk of bias for all domains. Four trials were sponsored by grants only, two trials by industry only, two trials by both grants and industry and three trials had an unknown funding source. Combined data from four trials (225 participants) suggested probiotics may reduce the number of pulmonary exacerbations during a four to 12 month time-frame, mean difference (MD) -0.32 episodes per participant (95% confidence interval (CI) -0.68 to 0.03; P = 0.07) (low-certainty evidence); however, the 95% CI includes the possibility of both an increased and a reduced number of exacerbations. Additionally, two trials (127 participants) found no evidence of an effect on the duration of antibiotic therapy during the same time period. Combined data from four trials (177 participants) demonstrated probiotics may reduce faecal calprotectin, MD -47.4 µg/g (95% CI -93.28 to -1.54; P = 0.04) (low-certainty evidence), but the results for other biomarkers mainly did not show any difference between probiotics and placebo. Two trials (91 participants) found no evidence of effect on height, weight or body mass index (low-certainty evidence). Combined data from five trials (284 participants) suggested there was no difference in lung function (forced expiratory volume at one second (FEV1) % predicted) during a three- to 12-month time frame, MD 1.36% (95% CI -1.20 to 3.91; P = 0.30) (low-certainty evidence). Combined data from two trials (115 participants) suggested there was no difference in hospitalisation rates during a three- to 12-month time frame, MD -0.44 admissions per participant (95% CI -1.41 to 0.54; P = 0.38) (low-certainty evidence). One trial (37 participants) reported health-related quality of life and while the parent report favoured probiotics, SMD 0.87 (95% CI 0.19 to 1.55) the child self-report did not identify any effect, SMD 0.59 (95% CI -0.07 to 1.26) (low-certainty evidence). There were limited results for gastrointestinal symptoms and intestinal microbial profile which were not analysable. Only four trials and one trial protocol (298 participants) reported adverse events as a priori hypotheses. No trials reported any deaths. One terminated trial (12 participants and available as a protocol only) reported a severe allergic reaction (severe urticaria) for one participant in the probiotic group. Two trials reported a single adverse event each (vomiting in one child and diarrhoea in one child). The estimated number needed to harm for any adverse reaction (serious or not) is 52 people (low-certainty evidence). AUTHORS' CONCLUSIONS: Probiotics significantly reduce faecal calprotectin (a marker of intestinal inflammation) in children and adults with CF, however the clinical implications of this require further investigation. Probiotics may make little or no difference to pulmonary exacerbation rates, however, further evidence is required before firm conclusions can be made. Probiotics are associated with a small number of adverse events including vomiting, diarrhoea and allergic reactions. In children and adults with CF, probiotics may be considered by patients and their healthcare providers. Given the variability of probiotic composition and dosage, further adequately-powered multicentre RCTs of at least 12 months duration are required to best assess the efficacy and safety of probiotics for children and adults with CF.

Gut Microbiota in Children With Cystic Fibrosis: A Taxonomic and Functional Dysbiosis.

Intestinal dysbiosis has been observed in children with cystic fibrosis (CF), yet the functional consequences are poorly understood. We investigated the functional capacity of intestinal microbiota and inflammation in children with CF. Stool samples were collected from 27 children with CF and 27 age and gender matched healthy controls (HC) (aged 0.8-18 years). Microbial communities were investigated by iTag sequencing of 16S rRNA genes and functional profiles predicted using Tax4Fun. Inflammation was measured by faecal calprotectin and M2-pyruvate kinase. Paediatric CF gastrointestinal microbiota demonstrated lower richness and diversity compared to HC. CF samples exhibited a marked taxonomic and inferred functional dysbiosis when compared to HC. In children with CF, we predicted an enrichment of genes involved in short-chain fatty acid (SCFA), antioxidant and nutrient metabolism (relevant for growth and nutrition) in CF. The notion of pro-inflammatory GI microbiota in children with CF is supported by positive correlations between intestinal inflammatory markers and both genera and functional pathways. We also observed an association between intestinal genera and both growth z-scores and FEV1%. These taxonomic and functional changes provide insights into gastrointestinal disease in children with CF and future gastrointestinal therapeutics for CF should explore the aforementioned pathways and microbial changes.

Differences in clinical outcomes of paediatric cystic fibrosis patients with and without meconium ileus.

BACKGROUND: Meconium ileus (MI) affects up to 20% of newborns with cystic fibrosis (CF). We compared clinical outcomes between Australian paediatric CF patients with and without meconium ileus (non-MI). METHODS: This was a retrospective case-control study of MI and non-MI patients in New South Wales, Australia, from 1988 to 2010. MI patients were matched 1:1 with pancreatic insufficient non-MI patients for age, sex and CF clinic. Clinical measurements, nutrition and gastrointestinal outcomes over this period were compared between groups using linear mixed models for continuous variables to account for age. RESULTS: There were 162 matched pairs (N=324, 52% female) with mean (SD) age of 15.3 (8.2) and 14.9 (7.9) years for MI and non-MI patients respectively (P=0.6). MI patients aged 5-23 had poorer FEV1% compared to non-MI patients (estimate -0.070 SE [0.02], P=0.003). There were no significant differences in P. aeruginosa isolation rates; however S. aureus isolation rates were lower in MI patients (72%) compared to non-MI (82%) (OR 0.6 [0.3-1.0], P=0.03). Chronic colonisation rates for P. aeruginosa and S. aureus were not significantly different between groups. MI patients aged 2-20 had significantly lower BMI Z-scores over time (estimate -0.25 SE [0.1], P=0.02). MI patients were more likely to receive oral feed supplements (OR 2.8 [1.4-6.1], P=0.003) and gastrostomy formation (OR 4.4 [1.1-24.6], P=0.02). CONCLUSIONS: CF patients with MI may have worse lung function, growth and nutrition than non-MI patients over time. Meconium ileus may be an early poor prognostic factor for CF.

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.

Protein O-fucosyltransferase 2-mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection.

Toxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease in immunocompromised individuals, and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity.

Aspartyl Protease 5 Matures Dense Granule Proteins That Reside at the Host-Parasite Interface in Toxoplasma gondii.

Toxoplasma gondii infects approximately 30% of the world's population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection, and several of these proteins are processed by the Golgi-associated aspartyl protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally derived peptides from wild-type and Δasp5 parasites. We reveal more than 2,000 unique Toxoplasma N-terminal peptides, mapping to both natural N termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterized ASP5 cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5 or in wild-type parasites following mutation of the motif from RRL to ARL. All identified ASP5 substrates are dense granule proteins, and interestingly, none appear to be exported, thus differing from the analogous system in related Plasmodium spp. Instead we show that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. We show that genetic deletion of WNG2 leads to attenuation in a mouse model, suggesting that this putative kinase is a new virulence factor in Toxoplasma Collectively, these data constitute the first in-depth analyses of ASP5 substrates and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.IMPORTANCEToxoplasma gondii is one of the most successful human parasites. Central to its success is the arsenal of virulence proteins introduced into the infected host cell. Several of these virulence proteins require direct maturation by the aspartyl protease ASP5, and all require ASP5 for translocation into the host cell, yet the true number of ASP5 substrates and complete repertoire of effectors is currently unknown. Here we selectively enrich N-terminally derived peptides using Terminal Amine Isotopic Labeling of Substrates (TAILS) and use quantitative proteomics to reveal novel ASP5 substrates. We identify, using two different enrichment techniques, new ASP5 substrates and their specific cleavage sites. ASP5 substrates include two kinases and one phosphatase that reside at the host-parasite interface, which are important for infection.

Protein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii.

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions.IMPORTANCEToxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

Age-related levels of fecal M2-pyruvate kinase in children with cystic fibrosis and healthy children 0 to 10years old.

BACKGROUND: The pathogenesis of gut inflammation, bacterial dysbiosis and increased rates of malignancy in CF is unclear. Fecal M2-pyruvate kinase (M2-PK) is a biomarker indicative of cellular proliferation that may be raised in intestinal malignancy and inflammation. Biomarkers, including M2-PK, may be useful in assessing effects of novel therapies on the gastrointestinal tract. METHODS: M2-PK was measured in stools collected from patients with CF and HC (0-10years). Linear mixed model analysis was used. RESULTS: M2-PK levels did not significantly change in children with CF (36 patients, 77 samples) (P=0.998) or HC (45 patients, 45 samples) (P=0.21), over the age range 0-10years. Patients with CF had elevated M2-PK compared to HC (median [IQR; range]: 10.7 [5.7-28.6; 1.0-239.1] (n=77) vs. 1.0 [1.0-1.0; 1.0-50.0] (n=45) U/mL, respectively; P=0.001). CONCLUSIONS: Fecal M2-PK was elevated in children with CF compared with HC during infancy and throughout childhood suggesting abnormalities in the CF gut exist in early life.

Age-dependent variation of fecal calprotectin in cystic fibrosis and healthy children.

BACKGROUND: Fecal calprotectin may be used as a non-invasive method to assess the effect of novel therapies on the gut in cystic fibrosis (CF). METHOD: Stools from CF patients and healthy controls (HC) (0-10years old) were prospectively collected for evaluation of temporal trends. RESULTS: 130 CF samples (64 subjects) and 114 HC samples (101 subjects) were collected. Overall, fecal calprotectin levels were different in CF patients and HC from 0 to 10years (P=0.0002). Fecal calprotectin in CF was significantly lower than HC from 0 to 1years (P=0.03) and demonstrated an upward trajectory until 4years. From >4 to 10years calprotectin was consistently higher in CF patients compared with HC (P=0.007). CONCLUSIONS: Fecal calprotectin levels in children with CF and HC were age-dependent and had distinct trajectories. Careful interpretation of calprotectin is required if used in drug trials for CF, particularly in children less than 4years old.

Differences in Outcomes between Early and Late Diagnosis of Cystic Fibrosis in the Newborn Screening Era.

OBJECTIVES: To evaluate children with cystic fibrosis (CF) who had a late diagnosis of CF (LD-CF) despite newborn screening (NBS) and compare their clinical outcomes with children diagnosed after a positive NBS (NBS-CF). STUDY DESIGN: A retrospective review of patients with LD-CF in New South Wales, Australia, from 1988 to 2010 was performed. LD-CF was defined as NBS-negative (negative immunoreactive trypsinogen or no F508del) or NBS-positive but discharged following sweat chloride < 60 mmol/L. Cases of LD-CF were each matched 1:2 with patients with NBS-CF for age, sex, hospital, and exocrine pancreatic status. RESULTS: A total of 45 LD-CF cases were identified (39 NBS-negative and 6 NBS-positive) with 90 NBS-CF matched controls. Median age (IQR) of diagnosis for LD-CF and NBS-CF was 1.35 (0.4-2.8) and 0.12 (0.03-0.2) years, respectively (P <.0001). Estimated incidence of LD-CF was 1 in 45 000 live births. Compared with NBS-CF, LD-CF had more respiratory manifestations at time of diagnosis (66% vs 4%; P <.0001), a higher rate of hospital admission per year for respiratory illness (0.49 vs 0.2; P = .0004), worse lung function (forced expiratory volume in 1 second percentage of predicted, 0.88 vs 0.97; P = .007), and higher rates of chronic colonization with Pseudomonas aeruginosa (47% vs 24%; P = .01). The LD-CF cohort also appeared to be shorter than NBS-CF controls (mean height z-score -0.65 vs -0.03; P = .02). CONCLUSIONS: LD-CF, despite NBS, seems to be associated with worse health before diagnosis and worse later growth and respiratory outcomes, thus providing further support for NBS programs for CF.