Non-coding RNAs: Emerging from the discovery to therapeutic applications.

The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.

New insights on molecular internalization and drug delivery following plasma jet exposures.

In this study, we evaluated cold atmospheric plasmas as a physical drug delivery tool for human cervical cancer HeLa cells and murine breast carcinoma 4T1 cells. Different cell exposure protocols - plasma jet, plasma treated medium, and combinations of plasma-induced electric field and plasma treated medium- have been proposed and assessed to provide new insight on plasma-induced uptake mechanism. Cell culture medium composition and volume are key parameters to achieve an efficient molecular uptake. The plasma device enabled the delivery of molecules having 150 kDa-size into 4T1cells. For the first time to our knowledge, substance uptake kinetics after plasma treatment were investigated. The percentage of positive cells for propidium iodide and an anti-cancer agent, doxorubicin, was higher when the drugs were added a few minutes after treatment. The Plasma treated medium was not found to be as efficient as direct plasma treatment in 4T1 cells while allowing an efficient delivery in HeLa cells. Uptake levels as high as 39.3 ±â€¯2.9% and 40.1 ±â€¯9.5% for HeLa and 4 T1 cells respectively were achieved for optimized operating conditions, for which the viability of the cells was not severely affected. We also observed that plasma treatment induced the formation of actin stress fibers into cells revealing a mechanical stress.

Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells.

Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Their proper delivery to dendritic cells (DCs) requires their protection against RNase degradation and more specificity for dose reduction. Lipid-Polymer-RNA lipopolyplexes (LPR) are attractive mRNA delivery systems and their equipment with mannose containing glycolipid, specific of endocytic receptors present on the membrane of DCs is a valuable strategy. In this present work, we evaluated the capacity of LPR functionalized with a tri-antenna of α-d-mannopyranoside (triMN-LPR) concerning (i) their binding to CD209/DC-SIGN and CD207/Langerin expressing cell lines, human and mouse DCs and other hematopoietic cell populations, (ii) the nature of induced immune response after in vivo immunization and (iii) their therapeutic anti-cancer vaccine efficiency. We demonstrated that triMN-LPR provided high induction of a local inflammatory response two days after intradermal injection to C57BL/6 mice, followed by the recruitment and activation of DCs in the corresponding draining lymph nodes. This was associated with skin production of CCR7 and CXCR4 at vaccination sites driving DC migration. High number of E7-specific T cells was detected after E7-encoded mRNA triMN-LPR vaccination. When evaluated in three therapeutic pre-clinical murine tumor models such as E7-expressing TC1 cells, OVA-expressing EG7 cells and MART-1-expressing B16F0 cells, triMN-LPR carrying mRNA encoding the respective antigens significantly exert curative responses in mice vaccinated seven days after initial tumor inoculation. These results provide evidence that triMN-LPR give rise to an efficient stimulatory immune response allowing for therapeutic anti-cancer vaccination in mice. This mRNA formulation should be considered for anti-cancer vaccination in Humans.

Effect of IL-1ß, TNF-α and IGF-1 on trans-endothelial passage of synthetic vectors through an in vitro vascular endothelial barrier of striated muscle.

When administrated in the blood circulation, plasmid DNA (pDNA) complexed with synthetic vectors must pass through a vascular endothelium to transfect underlying tissues. Under inflammatory condition, cytokines can modify the endothelium integrity. Here, the trans-endothelial passage (TEP) of DNA complexes including polyplexes, lipoplexes and lipopolyplexes was investigated in the presence of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) or insulin-like growth factor-1 (IGF-1). The experiments were performed by using an in vitro model comprising a monolayer of mouse cardiac endothelial cells (MCEC) seeded on a trans-well insert and the transfection of C2C12 myoblasts cultured on the lower chamber as read out of TEP. We report that polyplexes made with a histidinylated derivative of lPEI (His-lPEI) exhibit the highest capacity (10.5 µg cm-2 h versus 0.324 µg cm-2 h) to cross TNF-α-induced inflamed endothelium model, but this positive effect is counterbalanced by the presence of IL-1ß. His-lPEI polyplex TEP is also increased in the presence of IGF-1 (2.58 µg cm-2 h). TEP of lipid-based DNA complexes including lipoplexes and lipopolyplexes was lowest compared with polymer-based DNA complexes. Overall, the results indicate that under inflammation, His-lPEI polyplexes have a good profile to cross a vascular endothelium of striated muscle with low cytotoxicity and high transfection efficiency of C2C12 myoblasts. These data provide insights concerning the endothelial passage of vectors in inflammatory conditions and can serve as a basis towards in vivo studies.

[Guidelines for multivitamin administration in fortified human milk prepared for preterm infants]. / Protocole d'administration de l'Uvestérol ADEC® au prématuré nourri au lait de femme enrichi.

BACKGROUND: To reach nutritional standards, human milk has to have 2g/dL of protein. In 2013, Lafeber stated that when human milk is fortified up to 2g/dL, it may increase its osmolality up to 500 mOsm/kg. He also warned that care must be taken when adding a drug or vitamins to human milk. AIM: We studied, for the first time, the impact of adding multivitamins (ADEC) on human fortified milk osmolality. METHOD: The osmolality of 36 pasteurized, fortified human milk samples was measured. The amount of milk required as a solvent to maintain osmolality below 500 mOsm/kg was then determined. RESULTS: The osmolality of 2mL of fortified human milk reached up to 750 mOsm/kg when the multivitamins ADEC was added. The osmolality decreased proportionately as the solution was diluted and if vitamins are added in two half-doses each time. It is only with 20mL of milk that the osmolality lowers to its initial rate of 430 mOsm/kg. The stronger the milk's fortification is, the greater impact it has on the milk's osmolality. CONCLUSION: New nutritional recommendations for premature infants are needed. In the meantime, when the fortified milk intake is under 20mL, it is preferable to extend parenteral intakes with fat-soluble vitamins or reduce doses of vitamins in milk. Also, we should use enriched human milk as a fortifier and be cautious with indiscriminate fortification or when adding drugs and electrolyte solutions.

Evidence for plasmid DNA exchange after polyplex mixing.

The self-assembly of a plasmid DNA (pDNA) with cationic polymers or cationic liposomes forms nanosized supramolecular structures called lipoplexes, polyplexes and lipopolyplexes. Here, we report that when two polyplex preparations made using the same polymer and the same pDNA but labelled with two different fluorophores are mixed together, pDNA molecules are exchanged. Indeed, when Flu-pDNA complexed with histidinylated lPEI (Flu-pDNA/His-lPEI) polyplexes are mixed with Cy5-pDNA complexed with histidinylated lPEI (Cy5-pDNA/His-lPEI) polyplexes, a high quantity of polyplexes emitting dual fluorescence is observed and FRET indicates that one single polyplex contains two kinds of fluorescent pDNA molecules. This phenomenon depends on the polymer-type and the strength of the pDNA/polymer interaction. No exchange is observed with polylysine polyplexes, caged His-lPEI polyplexes, lipoplexes, lipopolyplexes or when His-lPEI polyplexes are mixed with lipoplexes. Our results suggest that aggregation or collapse of polyplexes occurs after their interaction leading to their unpackaging followed by the formation of new polyplexes with the exchange of pDNA.

Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia.

INTRODUCTION: Alcohol is known to decrease bone mineral density (BMD) and to induce trabecular microarchitecture deterioration. However, little is known about the effects of chronic alcohol consumption on osteocytes in situ. The aim of this study was to assess the effects of a high alcohol dose on osteocytes in an alcohol-induced osteopenia model. MATERIALS AND METHODS: 24 male Wistar rats, 2-months old were separated in 2 groups: Control (C) or Alcohol (A35). The rats in the A35 group drank a beverage composed of 35% ethanol v/v mixed to water for 17 weeks. BMD was assessed by DXA, while the microarchitecture was analyzed using µCT. Bone remodeling was studied measuring serum concentration of osteocalcin, NTx and TRAP. Bone marrow adiposity, osteoblastic lineage differentiation, osteocyte morphology and apoptosis were assessed using bright field, epifluorescence, transmission electron and confocal microscopy. RESULTS: BMD, trabecular thickness, TRAP and NTx concentration were significantly decreased in A35, while cortical thickness was thinner. There were 10 fold more cells stained with cleaved caspase-3, and 35% more empty lacunae in A35, these data indicating a large increase in osteocyte apoptosis in the A35 group. The number of lipid droplets in the marrow was increased in A35 (7 fold). Both the osteocyte apoptosis and the fat bone marrow content strongly correlated with femur BMD (p=0.0017, r = -0.72 and p=0.002, r = -0.70) and whole body BMD. CONCLUSION: These data suggest that low BMD is associated with osteocyte apoptosis and bone marrow fat content in alcohol-induced osteopenia.

Cold streams in early massive hot haloes as the main mode of galaxy formation.

Massive galaxies in the young Universe, ten billion years ago, formed stars at surprising intensities. Although this is commonly attributed to violent mergers, the properties of many of these galaxies are incompatible with such events, showing gas-rich, clumpy, extended rotating disks not dominated by spheroids. Cosmological simulations and clustering theory are used to explore how these galaxies acquired their gas. Here we report that they are 'stream-fed galaxies', formed from steady, narrow, cold gas streams that penetrate the shock-heated media of massive dark matter haloes. A comparison with the observed abundance of star-forming galaxies implies that most of the input gas must rapidly convert to stars. One-third of the stream mass is in gas clumps leading to mergers of mass ratio greater than 1:10, and the rest is in smoother flows. With a merger duty cycle of 0.1, three-quarters of the galaxies forming stars at a given rate are fed by smooth streams. The rarer, submillimetre galaxies that form stars even more intensely are largely merger-induced starbursts. Unlike destructive mergers, the streams are likely to keep the rotating disk configuration intact, although turbulent and broken into giant star-forming clumps that merge into a central spheroid. This stream-driven scenario for the formation of discs and spheroids is an alternative to the merger picture.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta-adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrbeta2R and the absence of Adrbeta1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (-52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Young's Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrbeta2R-deficient mice, suggest that low doses of beta-blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues.

[Sonopermeabilization: therapeutic alternative with ultrasound and microbubbles]. / Sonoperméabilisation: alternative thérapeutique par ultrasons et microbulles.

Future applications of ultrasound and microbubbles extend to more than imaging applications. Over the last few years, it was reported that sonographic contrast agent effects under ultrasound, modulate transiently cell membrane permeability. This process, named sonoporation and classified as a new physical method to transfer genes or drugs, consists of using a physical energy source to modulate membrane integrity. The possibility to transfer therapeutic genes would be a new tool for gene therapy and could constitute an alternative method. After in vitro and in vivo studies presentation, the therapeutic potential of sonoporation will be investigated in this paper.

mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes.

Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route. Synthetic melanoma-associated antigen MART1 mRNA was formulated with a polyethylene glycol (PEG)ylated derivative of histidylated polylysine and L-histidine-(N,N-di-n-hexadecylamine)ethylamide liposomes (termed histidylated lipopolyplexes). Lipopolyplexes comprised mRNA/polymer complexes encapsulated by liposomes. The tumor protective effect was induced with MART1 mRNA carrying a poly(A) tail length of 100 adenosines at an optimal dose of 12.5 microg per mouse. MART1 mRNA lipopolyplexes elicited a cellular immune response characterized by the production of interferon-gamma and the induction of cytotoxic T lymphocytes. Finally, the anti-B16 response was enhanced using a formulation containing both MART1 mRNA and MART1-LAMP1 mRNA encoding the antigen targeted to the major histocompatibility complex class II compartments by the lysosomal sorting signal of LAMP1 protein. Our results provide a basis for the development of mRNA histidylated lipopolyplexes for cancer vaccine.

Lattice melting and rotation in perpetually pulsating equilibria.

Systems whose potential energies consists of pieces that scale as r;{-2} together with pieces that scale as r;{2} , show no violent relaxation to Virial equilibrium but may pulsate at considerable amplitude forever. Despite this pulsation these systems form lattices when the nonpulsational "energy" is low, and these disintegrate as that energy is increased. The "specific heats" show the expected halving as the "solid" is gradually replaced by the "fluid" of independent particles. The forms of the lattices are described here for N18 and they become hexagonal close packed for large N . In the larger N limit, a shell structure is formed. Their large N behavior is analogous to a gamma=53 polytropic fluid with a quasigravity such that every element of fluid attracts every other in proportion to their separation. For such a fluid, we study the "rotating pulsating equilibria" and their relaxation back to uniform but pulsating rotation. We also compare the rotating pulsating fluid to its discrete counterpart, and study the rate at which the rotating crystal redistributes angular momentum and mixes as a function of extra heat content.

[Prenatal screening of cytomegalovirus in France: an increasing frequency practices of screening from 2000 to 2003 despite no recommendation in favor]. / Dépistage prénatal du cytomégalovirus en France: une augmentation des pratiques de prescription de 2000 à 2003 malgré l'absence de recommandation favorable.

BACKGROUND: Screening cytomegalovirus infection in pregnant women is still controversial in 2004 in France. In this context, we evaluated the interest of such a screening in 2004 in France. This paper was designed to describe trends in CMV prenatal screening practices in 2000-2003 in France. METHODS: This retrospective study, describes the prescription of CMV screening in HIV-negative pregnant women giving birth in the private care sector, according to their occupational category and geographical area. Data were provided by the "Caisse d'Assurance-maladie des Travailleurs Indépendants" (independent workers health insurance fund). RESULTS: The study included 34.347 women, delivering in 2001-2004 (beginning of pregnancy in 2000-2003). The number of pregnant women screened for CMV increased significantly between 2000 (5.8%, 301/5.177), 2001 (11.1%, 1.130/10.139) and 2002 (22.1%, 2.701/12.223), (p<0.001), then was stable in 2003 (22.0%, 1.496/6.808). The percentage of women screened for CMV, at least once during pregnancy, doubled between 2001 and 2002 (p<0.001) in each occupational category and geographical area. It was significantly different between occupational categories (p<0.01), with a higher percentage of women in the self-employed and commercial agent occupational categories than in the craftsman category. There was also a significant difference between geographical areas (p<0.001), with a higher rate in Paris. CONCLUSION: This study providing baseline information on CMV practices showed: 1- a significant increase in the frequency of CMV screening among pregnant women over the period 2000-2002 with a stabilization in 2003; 2- a similar trend observed in each occupation category and geographical area but with a markedly higher frequency of screening practices in the Paris area and among self-employed women. A study measuring the effect of the 2004 ANAES recommendation suggesting not to screen for CMV during pregnancy should be conducted.

Non-viral vectors in cystic fibrosis gene therapy: progress and challenges.

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Histidine-rich peptides and polymers for nucleic acids delivery.

Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes. Histidylated polylysine forms cationic particles of 100 nm with a plasmid and yielded a transfection of 3-4.5 orders of magnitude higher than polylysine. The biological activity of antisense ODN was increased more than 20-fold when it was complexed with highly histidylated oligolysine into small cationic spherical particles of 35 nm. Evidence that imidazole protonation mediates the effect of these molecules in endosomes are provided. We also describe a disulfide-containing polylysine conjugate capable of mediating DNA unpackaging in a reductive medium and to increase the transfection efficiency. Overall, these molecules constitute interesting devices for developing non-viral gene delivery systems.