Nutritional Control of L1 Arrest and Recovery in Caenorhabditis elegans by Insulin-like Peptides and Signaling

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.

Polymorphisms in the kinesin-like factor 1 B gene and risk of epithelial ovarian cancer in Eastern Chinese women.

The kinesin-like factor 1 B (KIF1B) gene plays an important role in the process of apoptosis and the transformation and progression of malignant cells. Genetic variations in KIF1B may contribute to risk of epithelial ovarian cancer (EOC). In this study of 1,324 EOC patients and 1,386 cancer-free female controls, we investigated associations between two potentially functional single nucleotide polymorphisms in KIF1B and EOC risk by the conditional logistic regression analysis. General linear regression model was used to evaluate the correlation between the number of variant alleles and KIF1B mRNA expression levels. We found that the rs17401966 variant AG/GG genotypes were significantly associated with a decreased risk of EOC (adjusted odds ratio (OR) = 0.81, 95 % confidence interval (CI) = 0.68-0.97), compared with the AA genotype, but no associations were observed for rs1002076. Women who carried both rs17401966 AG/GG and rs1002076 AG/AA genotypes of KIF1B had a 0.82-fold decreased risk (adjusted 95 % CI = 0.69-0.97), compared with others. Additionally, there was no evidence of possible interactions between about-mentioned co-variants. Further genotype-phenotype correlation analysis indicated that the number of rs17401966 variant G allele was significantly associated with KIF1B mRNA expression levels (P for GLM = 0.003 and 0.001 in all and Chinese subjects, respectively), with GG carriers having the lowest level of KIF1B mRNA expression. Taken together, the rs17401966 polymorphism likely regulates KIF1B mRNA expression and thus may be associated with EOC risk in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection.

DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to. Finally, cell type specific footprints were detected within DNase hypersensitive sites that are present in multiple cell types, further supporting that footprints can identify changes in TF binding that are not detectable using other strategies.

Drug-drug interaction studies of cardiovascular drugs involving P-glycoprotein, an efflux transporter, on the pharmacokinetics of edoxaban, an oral factor Xa inhibitor.

BACKGROUND: Edoxaban, an oral direct factor Xa inhibitor, is in development for thromboprophylaxis, including prevention of stroke and systemic embolism in patients with atrial fibrillation (AF). P-glycoprotein (P-gp), an efflux transporter, modulates absorption and excretion of xenobiotics. Edoxaban is a P-gp substrate, and several cardiovascular (CV) drugs have the potential to inhibit P-gp and increase drug exposure. OBJECTIVE: To assess the potential pharmacokinetic interactions of edoxaban and 6 cardiovascular drugs used in the management of AF and known P-gp substrates/inhibitors. METHODS: Drug-drug interaction studies with edoxaban and CV drugs with known P-gp substrate/inhibitor potential were conducted in healthy subjects. In 4 crossover, 2-period, 2-treatment studies, subjects received edoxaban 60 mg alone and coadministered with quinidine 300 mg (n = 42), verapamil 240 mg (n = 34), atorvastatin 80 mg (n = 32), or dronedarone 400 mg (n = 34). Additionally, edoxaban 60 mg alone and coadministered with amiodarone 400 mg (n = 30) or digoxin 0.25 mg (n = 48) was evaluated in a single-sequence study and 2-cohort study, respectively. RESULTS: Edoxaban exposure measured as area under the curve increased for concomitant administration of edoxaban with quinidine (76.7 %), verapamil (52.7 %), amiodarone (39.8 %), and dronedarone (84.5 %), and exposure measured as 24-h concentrations for quinidine (11.8 %), verapamil (29.1 %), and dronedarone (157.6 %) also increased. Administration of edoxaban with amiodarone decreased the 24-h concentration for edoxaban by 25.7 %. Concomitant administration with digoxin or atorvastatin had minimal effects on edoxaban exposure. CONCLUSION: Coadministration of the P-gp inhibitors quinidine, verapamil, and dronedarone increased edoxaban exposure. Modest/minimal effects were observed for amiodarone, atorvastatin, and digoxin.