Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct.

Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Genome-enabled analysis of population dynamics and virulence associated loci in the oat crown rust fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the USA and South Africa (SA). Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the USA and SA. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the USA and contributions from clonality and sexuality. The population from SA is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define eleven virulence-associated loci corresponding to twenty-five oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the USA to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognising genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended.

Genomics accelerated isolation of a new stem rust avirulence gene-wheat resistance gene pair.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat-rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.

Evaluation of neurobehavioral abnormalities and immunotoxicity in response to oral imidacloprid exposure in domestic chickens (Gallus gallus domesticus).

Domestic chickens (Gallus gallus domesticus) were exposed to imidacloprid by gavage once daily for 7 consecutive days at 0, 0.03, 0.34, 3.42, 10.25, and 15.5 mg/kg/day (n = 20 per group; 5 6-week-old males, 5 6-week-old females, 5 9-week-old males, and 5 9-week-old females). The severity and duration of neurobehavioral abnormalities were recorded. Components of the innate and adaptive immune system were assessed with 7 standard functional assays. Temporary neurobehavioral abnormalities were observed in a dose-dependent manner, including muscle tremors, ataxia, and depressed mentation. Based upon mean clinical severity scores, the no observed adverse effect level (NOAEL) was 3.42 mg/kg/day, and the lowest observed adverse effect level (LOAEL) was 10.25 mg/kg/day. The effective dose value for the presence of any neurobehavioral abnormalities in 50% of the test group (ED50) was 4.62 ± 0.98 mg/kg/day. The ED50 for an adjusted score that included both severity and duration of neurobehavioral abnormalities was 11.24 ± 9.33 mg/kg/day. These ED50 values are equivalent to a 1 kg bird ingesting 29 or 70 imidacloprid treated soybean seeds respectively. Immunotoxicity was not documented, possible causes include the assays were insensitive, relevant immune functions were not examined, or imidacloprid is not immunotoxic at this dosing schedule in this species. Neurobehavioral abnormalities were a more sensitive indicator of the sublethal effects of imidacloprid than immunotoxicity.

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation.

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

Genetic structure in Red Junglefowl (Gallus gallus) populations: Strong spatial patterns in the wild ancestors of domestic chickens in a core distribution range.

Red Junglefowl (Gallus gallus) are among the few remaining ancestors of an extant domesticated livestock species, the domestic chicken, that still occur in the wild. Little is known about genetic diversity, population structure, and demography of wild Red Junglefowl in their natural habitats. Extinction threats from habitat loss or genetic alteration from domestic introgression exacerbate further the conservation status of this progenitor species. In a previous study, we reported extraordinary adaptive genetic variation in the MHC B-locus in wild Red Junglefowl and no evidence of allelic introgression between wild and domestic chickens was observed. In this study, we characterized spatial genetic variation and population structure in naturally occurring populations of Red Junglefowl in their core distribution range in South Central Vietnam. A sample of 212 Red Junglefowl was obtained from geographically and ecologically diverse habitats across an area of 250 × 350 km. We used amplified fragment-length polymorphism markers obtained from 431 loci to determine whether genetic diversity and population structure varies. We found that Red Junglefowl are widely distributed but form small and isolated populations. Strong spatial genetic patterns occur at both local and regional scales. At local scale, population stratification can be identified to approximately 5 km. At regional scale, we identified distinct populations of Red Junglefowl in the southern lowlands, northern highlands, and eastern coastal portions of the study area. Both local and long-distance genetic patterns observed in wild Red Junglefowl may reflect the species' ground-dwelling and territorial characteristics, including dispersal barriers imposed by the Annamite Mountain Range. Spatially explicit analyses with neutral genetic markers can be highly informative and here elevates the conservation profile of the wild ancestors of domesticated chickens.

Genetic variation of major histocompatibility complex (MHC) in wild Red Junglefowl (Gallus gallus).

The major histocompatibility complex (MHC) is a multi-family gene cluster that encodes proteins with immuno-responsive function. While studies of MHC in domesticated poultry are relatively common, very little is known about this highly polymorphic locus in wild Red Junglefowl (Gallus gallus), the natural progenitor of domestic chickens. We investigated the diversity of MHC within and among four wild Red Junglefowl populations across diversified natural habitats in South Central Vietnam. Based on a SNP panel of 84 sites spanning 210 Kb of the MHC-B locus, we identified 310 unique haplotypes in 398 chromosomes. None of these haplotypes have been described before and we did not observe any of the wild Red Junglefowl haplotypes in domesticated chickens. Analysis of molecular variance (AMOVA) revealed that 94.51% of observed haplotype variation was accounted for at the within individual level. Little genetic variance was apportioned within and among populations, the latter accounting only for 0.83%. We also found evidence of increased recombination, including numerous hotspots, and limited linkage disequilibrium among the 84 SNP sites. Compared to an average haplotype diversity of 3.55% among seventeen lines of domestic chickens, our results suggest extraordinarily high haplotype diversity remains in wild Red Junglefowl and is consistent with a pattern of balancing selection. Wild Red Junglefowl in Vietnam, therefore, represent a rich resource of natural genomic variation independent from artificial selection.