The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.

Objective: Examine the accuracy of parental weight perceptions of overweight children before and after the implementation of childhood obesity legislation that included BMI screening and feedback. Methods and Procedures: Statewide telephone surveys of parents of overweight (BMI ≥ 85th percentile) Arkansas public school children before (n = 1,551; 15% African American) and after (n = 2,508; 15% African American) policy implementation were examined for correspondence between parental perception of child's weight and objective classification. Results: Most (60%) parents of overweight children underestimated weight at baseline. Parents of younger children were significantly more likely to underestimate (65%) than parents of adolescents (51%). Overweight parents were not more likely to underestimate, nor was inaccuracy associated with parental education or socioeconomic status. African-American parents were twice as likely to underestimate as whites. One year after BMI screening and feedback was implemented, the accuracy of classification of overweight children improved (53% underestimation). African-American parents had significantly greater improvements than white parents (P < 0.0001). Discussion: Parental recognition of childhood overweight may be improved with BMI screening and feedback, and African-American parents may specifically benefit. Nonetheless, underestimation of overweight is common and may have implications for public health interventions.

Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.

SUMMARY piRNAs silence foreign genes, such as transposons, to preserve genome integrity, but also target endogenous mRNAs by mechanisms that are poorly understood. C. elegans piRNAs interact with both transposon and non-transposon mRNAs to initiate sustained silencing via the RNAi pathway. To assess the dysregulation of gene silencing caused by lack of piRNAs, we restored RNA silencing in RNAi-defective animals in the presence or absence of piRNAs. In the absence of piRNAs and a cellular memory of piRNA activity, essential and conserved genes are misrouted into the RNAi pathway to produce siRNAs that bind the nuclear Argonaute HRDE-1, resulting in dramatic defects in germ cell proliferation and function such that the animals are sterile. Inactivation of RNAi suppresses sterility, indicating that aberrant siRNAs produced in the absence of piRNAs target essential genes for silencing. Thus, by reanimating RNAi we uncovered a role for piRNAs in protecting essential genes from RNA silencing.

Dispersal of pollen, seeds, or vegetative propagules from intensively bred, exotic, or recombinant DNA modified forest plantations may cause detrimental or beneficial ecological impacts on wild or managed ecosystems. Insertion of genes designed to prevent or substantially reduce dispersal could reduce the risk and extent of undesired impacts. Containment measures may also be required by law or marketplace constraints, regardless of risks or benefits. We discuss: (1) the context for when genetic containment or mitigation systems may be needed;(2) technology approaches and mechanisms; (3) the state of knowledge on genes/genomics of sexual reproduction in forest trees; (4) stability of transgene expression during vegetative growth; (5) simulation studies to define the level of containment needed; and (6) needed research to deliver effective containment technologies. We illustrate progress with several examples from our research on recombinant DNA modified poplars. Our simulations show that even partial sterility can provide very substantial reductions in gene flow into wild trees. We conclude that it is impossible to define the most effective containment approaches, nor their reliability, based on current genomic knowledge and technological tools. Additional genomic and technological studies of a wide variety of options are needed. Studies in field environments are essential to provide data relevant to ecological analysis and regulatory decisions and need to be carried out in phylogenetically diverse representatives of the economically most important taxa of forest trees.

Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with most miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline where it localizes alongside the piRNA and siRNA machinery at P granules. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline.

Summary More than two thousand C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized siRNA amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells [1]. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNA interference. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes, however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation, mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant negative missense mutant of MUT-14 is RNAi-defective in vivo, however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.

Mediterranean annual grasses have invaded California and have replaced vast areas of native grassland. One of these invasive grasses is Brachypodium distachyon, a new model species for the grasses with extensive genomic resources and a nearly completed genome sequence. This study shows that the level of genetic variation in invaded California grasslands is lower compared to the native range in Eurasia. The invaded regions are characterized by highly differentiated populations of B. distachyon isolated by distance, most likely as a result of founder effects and a dearth of outcrossing events. EXP6 and EXP10 encoding alpha-expansins responsible for rapid growth, and AGL11 and AGL13 encoding proteins involved in vegetative phase regulation, appear to be under purifying selection with no evidence for local adaptation. Our data show that B. distachyon has diverged only recently from related Brachypodium species and that tetraploidization might have been as recent as a few thousand years ago. Observed low genetic variation in EXP10 and AGL13 appears to have been present in Eurasia before tetraploidization, potentially as a result of strong selective pressures on advantageous mutations, which are most likely responsible for its fast growth and rapid completion of its life cycle.