Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms

Slaughter weight rather than sex affects carcass cuts and tissue composition of Bisaro pigs

Carcass cuts and tissue composition were assessed in Bisaro pigs (n=64) from two sexes (31 gilts and 33 entire males) reared until three target slaughter body-weights (BW) means: 17 kg, 32 kg, and 79 kg. Dressing percentage and backfat thickness increased whereas carcass shrinkage decreased with increasing BW. Slaughter weight affected most of the carcass cut proportions, except shoulder and thoracic regions. Bone proportion decreased linearly with increasing slaughter BW, while intermuscular and subcutaneous adipose tissue depots increased concomitantly. Slaughter weight increased the subcutaneous adipose tissue proportion but this impaired intramuscular and intermuscular adipose tissues in the loin primal. The sex of the pigs minimally affected the carcass composition, as only the belly weight and the subcutaneous adipose tissue proportions were greater in gilts than in entire males. Light pigs regardless of sex are recommended to balance the trade-offs between carcass cuts and their non-edible compositional outcomes.Work included in the Portuguese PRODER research Project BISOPORC – Pork extensive production of Bísara breed, in two alternative systems: fattening on concentrate vs chesnut, Project PRODER SI I&DT Medida 4.1 “Cooperação para a Inovação”. The authors are grateful to Laboratory of Carcass and Meat Quality of Agriculture School of Polytechnic Institute of Bragança ‘Cantinho do Alfredo’. The authors are members of the MARCARNE network, funded by CYTED (ref. 116RT0503).info:eu-repo/semantics/publishedVersio

Potential Role of AGR2 for Mammalian Skin Wound Healing

The limited ability of mammals to regenerate has garnered significant attention, particularly in regard to skin wound healing (WH), which is a critical step for regeneration. In human adults, skin WH results in the formation of scars following injury or trauma, regardless of severity. This differs significantly from the scarless WH observed in the fetal skin of mammals or anamniotes. This review investigates the role of molecular players involved in scarless WH, which are lost or repressed in adult mammalian WH systems. Specifically, we analyze the physiological role of Anterior Gradient (AGR) family proteins at different stages of the WH regulatory network. AGR is activated in the regeneration of lower vertebrates at the stage of wound closure and, accordingly, is important for WH. Mammalian AGR2 is expressed during scarless WH in embryonic skin, while in adults, the activity of this gene is normally inhibited and is observed only in the mucous epithelium of the digestive tract, which is capable of full regeneration. The combination of AGR2 unique potencies in postnatal mammals makes it possible to consider it as a promising candidate for enhancing WH processes

Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate

We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism

Images of tadpole heads as seen from the dorsal side and the sequences of MO, pHypNA and HypNA-pPNA oligomers used in mRNA translation inhibitory assays

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The injection of antisense oligomers results in severe abnormalities of the head structure. The side of injection is shown by red arrow. () A control tadpole head; ( and ) heads of tadpoles developed from the embryos microinjected with the antisense MO, pHypNA and HypNA-pPNA oligomers, respectively; ( and ) heads of tadpoles developed from the embryos microinjected with the mismatched pHypNA and HypNA-pPNA oligomers

Cellular uptake and intracellular distribution of the fluorescein-labeled mimic oligomers (TCACTCAACACTCAC-Flu)

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Fluorescent analysis of the delivery of oligomers (1 µM) into Phoenix Eco cells performed 20 h after the treatment under various delivery conditions: in the presence, or in the absence, of LFA, 6 mM CaCl, and 100 µM chloroquine (ClQ). () Confocal microscopy images of the pHypNA oligomer uptake after 20 h incubated with unfixed Phoenix Eco cells in the presence of: LFA (1); LFA/6mM Ca (2); CT-ODN/LFA (3) and LFA/100 µM ClQ (4)

Dose-dependent inhibition of firefly luciferase translation by the mimic antisense oligomers targeted against the translational start site of FLuc mRNA

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Sequences of HypNA-pPNA and pHypNA oligomers designed to target the FLuc mRNA; () Analysis of firefly luciferase translation inhibition by the oligomers. Firefly luciferase activity was calculated relative to the activity in the absence of any oligomer and normalized respect to luciferase production

Analysis of the stability of the fluorescein-labeled pHypNA oligomer (Flu-1) by the electrophoresis in a 15% polyacrylamide gel in denaturing conditions

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The oligomer (5 µM) was incubated at 37°C in 500 µl of DMEM/10% FBS. Aliquots (20 µl) were taken at each 24 h and frozen at −20°C until analysis by electrophoresis. () The image shows probes of the mimic oligomer taken after 48 h (1), 72 h (2), 120 h (3) and 0 h (4) incubation; the visualization was in UV-light at 365 nm. () The comparison of the stability of the fluorescein-labeled pHypNA oligomer and the control 3′-fluorescein-labeled oligodeoxyribonucleotide of the same sequence. Densitometric calculations of the amount of full-length oligomer were done using UVP Bio-Imaging system and LabWorks imaging software

Analysis of the reduction of TurboGFP levels by antisense mimic oligomers in Phoenix Eco cells transfected with p2FP-RNAi vector

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Co-transfection of oligomers was performed in the presence of LFA. The cells transformed with the vector alone were analyzed as controls. () Sequences of oligomers designed to target the translational start site of the TurboGFP mRNA. () Analysis of green and red fluorescence intensity of the cells treated with 0.5 µM oligomers, or with 0.2 µM dsRNA, after 24 h. The average data of three separate experiments are shown. () Fluorescent microscopy images of the cells transfected with p2FP-RNAi and treated with 0.5 µM mimic oligomer samples for 16 and 24 h. Panels show examples of cells treated with 0.2 µM dsRNA (a); mismatched pHypNA oligomer (b); duplex of antisense pHypNA oligomer with CT-ODN (c); duplex of antisense HypNA-pPNA oligomer with CT-ODN (d); antisense pHypNA oligomer (e) and antisense HypNA-pPNA oligomer (f). Panel (g) shows cells untreated with oligomers. () Effect of the antisense oligomer concentration on the TurboGFP production in cells. Fluorescence was measured 24 h after the transfection. Average data points from three independent experiments are shown