How does a juvenile insect transform into an adult? This question, which sums up the wonder of insect metamorphosis, has fascinated mankind since ancient times. Modern physiology has established the endocrine basis regulating these transformations, which mainly depend on two hormone types: ecdysteroids, which promote molts, and juvenile hormones, which repress the transformation into the adult stage. The interplay of these two hormones regulates the genes involved in juvenile and adult programs and the shift from one to the other. microRNAs (miRNAs) are small noncoding RNAs, which participate in many biological processes, and we wondered whether they might be also involved in insect metamorphosis. In insects, Dicer-1 ribonuclease transforms miRNA precursors into mature miRNAs. Thus, using systemic RNA interference (RNAi) to silence the expression of Dicer-1 in the hemimetabolan insectBlattella germanica, we depleted miRNA contents in the last instar nymph. This practically inhibited metamorphosis after the next molt, as the resulting specimens showed nymphoid features and were able to molt again. The experiments show that miRNAs play a key role in hemimetabolan metamorphosis, perhaps regulating genes that are juvenile hormone targets.

Quercetin is a major flavonoid in the human diet and the most commonly used in studies of biological activity. Most of the knowledge about its biological effects has originated from in vitro studies while in vivo data are scarce. Quercetin mostly occurs in foodstuffs as glycosides that are deglycosylated during absorption and further submitted to different conjugation reactions. Methylation to isorhamnetin (quercetin 3′-O-methylether) or tamarixetin (quercetin 4′-O-methylether) seems to be an important conjugation process in quercetin metabolism. In this work, the effects of quercetin and its 3′- and 4′-O-methylated metabolites on the phenotypic characteristics, stress oxidative resistance, thermotolerance and lifespan of the model organism Caenorhabditis elegans have been assessed. The three assayed flavonols significantly prolonged the lifespan of this nematode with an increase from 11% to 16% in the mean lifespan with respect to controls. However, only quercetin significantly increased the reproductive capacity of the worm and enlarged the body size. Exposure to the assayed flavonols also increased significantly the resistance against thermal and juglone-induced oxidative stress, although differences were found depending on the stage of development of the worm. Thus, quercetin offered greater protection when thermal stress was applied in the 1st day of adulthood, whereas tamarixetin was more efficient in worms submitted to stress in the 6th day of adulthood. Similarly, significantly greater protection was provided by quercetin than by its methylated derivatives at the 1st day of adulthood, whilst quercetin and isorhamnetin were equally efficient when the oxidative stress was induced in the 6th of day of adulthood. Further evidence of antioxidant protection was obtained checking the oxidation status of proteins by the OxyBlot™ detection kit. Analyses by HPLC-DAD-ESI/MS confirmed that the three flavonols were taken up by C. elegans leading to the formation of some glycosylated, sulfated and methylated metabolites, and that demethylation of these latter to quercetin was also produced. Quantification of the levels of quercetin, isorhamnetin and tamarixetin, as well as their detected metabolites indicated a greater uptake of quercetin than its methylated derivatives by the nematode.

Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation. The gene transcription profile determines the developmental state of an organism. During embryogenesis, aging, starvation or any lifecycle stage, organisms express specific sets of genes that must be turned off at other stages to maintain the correct metabolic and differentiated state of the cells. Mutations that disrupt control of signaling pathways may give rise to certain types of tumors. This happens with mutations in genes coding for the Integrator complex, a multi-protein complex involved in the processing of small nuclear RNAs (snRNAs). Here, we uncover a major mechanism underlying gene expression changes in mutants affecting the Integrator complex. Using a Caenorhabditis elegans model, we describe how the lack of snRNA processing leads to transcription of genes located downstream of the snRNA loci. This primary alteration is not restricted only to those genes but has a broad effect on the expression of other genes involved in the regulation of signaling pathways by protein phospho-modification.

Objectives: Achieving targeted delivery of gene medicines is desirable to maximise activity. Here, galactosylated amphiphilic cyclodextrins (CDs) are examined in terms of their ability to transfect asialoglycoprotein receptor-bearing HepG2 cells. Methods: Cationic amphiphilic CDs were synthesised as well as amphiphilic CDs bearing galactose-targeting ligands with different linker lengths. Binding of galactosylated CDs to a galactose-specific lectin was examined by surface plasmon resonance. CDs were formulated with and without the helper lipid DOPE and complexed with plasmid DNA. Transfection was evaluated by luciferase assay. Intracellular trafficking was assessed by confocal microscopy. Key findings: Binding of targeted CDs to a galactose-specific lectin was achieved. Binding decreased with linker length between the galactosyl group and the CD core. Contrary to the lectin binding results, transfection levels increased with an increase in linker length from 7 atoms to 15. Compared to non-targeted formulations, a significant increase in transfection was observed only in the presence of the helper lipid DOPE. Confocal microscopy revealed that DOPE caused a pronounced effect on cellular distribution. Conclusions: The galactose-targeting ligand induced substantial increases in transfection over non-targeted formulations when DOPE was included, indicating the potential for targeted gene delivery using CD-based delivery systems.