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How is ownership established over non-owned things? We suggest that people may view ownership as a kind of credit given to agents responsible for making possession of a non-owned object possible. On this view, judgments about the establishment of ownership depend on attributions of responsibility. We report three experiments showing that people's judgments about the establishment of ownership are influenced by an agent's intent and control in bringing about an outcome, factors that also affect attributions of responsibility. These findings demonstrate that people do not just consider who was first to possess an object in judging who owns it, and are broadly consistent with the view that ownership is acquired through labor. The findings also suggest that rather than exclusively being the product of social conventions, judgments about the establishment of ownership over non-owned things also depend on the psychological processes underlying the attribution of responsibility. (Contains 3 figures and 1 supplementary data.)

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor (1,2). Nf1 encodes neurofibromin, a GTPaseactivating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38a. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance., Aberrant Ras signalling contributes to the pathogenesis of myeloid malignancies and can result from acquired RAS mutations or from alternative genetic mechanisms that include FLT3 internal tandem duplications, the BCR-ABL [...]

Recent research has shown that infants selectively approach prosocial versus antisocial characters, suggesting that foundations of sociomoral development may be present early in life. Despite this, to date, the mental processes involved in infants’ prosocial preferences are poorly understood. To explore a possible role of emotions in early social evaluations, the current studies examined whether four samples of infants and toddlers express different emotional reactions after observing prosocial (giving) versus antisocial (taking) events. Experimentally blind coders rated infants’ and toddlers’ emotional reactions to prosocial and antisocial interactions from video using a 1‐ to 7‐point Likert scale of negative to positive emotion; reactions were rated as more positive after viewing prosocial compared to antisocial interactions in three of four samples. While the observed effects were small, a single‐paper meta‐analysis suggests that the findings are robust and stable across age. These results support the possibility that emotional reactions play some role in infants’ sociomoral evaluations. Statement of contribution What is already known Infants prefer prosocial to antisocial individuals from the first year of life. Emotion plays some role in the sociomoral judgments of children and adults. What this study adds Infants and toddlers express more positive reactions after observing prosocial giving versus antisocial taking acts, though observed effect sizes are small. Naïve coders can predict at a better than chance rate what type of act an infant or toddler just viewed based on their facial expressions. Provides the first evidence that emotion plays some to‐be‐specified role in infants’ and toddlers’ sociomoral evaluations. [ABSTRACT FROM AUTHOR]

Abstract: In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly(iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN. [Copyright &y& Elsevier]

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4°C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25kDa polyethylenimine (PEI) as a carrier. After 2.5h of incubation at 37°C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular Distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A... [ABSTRACT FROM AUTHOR]

Clinical evaluation of the genetic testing strategy is essential for ensuring the correct determination of mutation carriers. The current study retrospectively analyzed genetic and clinicopathological data from 62 Vietnamese patients with retinoblastoma (RB) referred to the Vinmec Hi-Tech Center for RB transcriptional corepressor 1 ( RB1 ) genetic testing between 2017 and 2019. The present study aimed to evaluate the sensitivity of the Next Generation Sequencing (NGS) method to identify novel RB1 mutations, and to consider using age at diagnosis as a risk factor. Genomic DNA was analyzed with custom panel based targeted NGS. NGS was performed on the Beijing Genomics Institute (BGI) sequencing platform, and pathogenic or likely pathogenic variants were confirmed by Sanger sequencing, quantitative PCR (qPCR) or Multiplex Ligation-dependent Probe Amplification assay (MLPA). Constitutional RB1 variants were identified in 100% (25/25) of the bilateral cases, while several common previously reported RB1 mutations were also recorded. In addition, in Vietnamese patients with RB, nine novel RB1 mutations were identified. Children aged between 0-36 months were more likely to be RB1 carriers compared with those aged >36 months. The current findings indicated that the NGS method implemented in the Vinmec Hi-Tech Center was highly accurate, and age at diagnosis may be used to assess the risk of hereditary RB. Furthermore, the newly identified RB1 mutations may provide additional data to improve the current understanding of the mechanisms underlying RB1 inactivation and the development of rapid assays for detecting RB1 mutations. Overall, the present study suggested that NGS may be applied for detecting germline RB1 mutations in routine clinical practice., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020, Spandidos Publications.)

One of the main limitations in the use of nucleoside reverse transcriptase inhibitors (NRTIs) such as azidothymidine (AZT) lies in their poor intracellular activation by cellular kinases into their active tri-phosphorylated form. Thus, the direct administration of triphosphate NRTIs like azidothymidine-triphosphate (AZT-TP), has been considered for bypassing this metabolic bottleneck, but these molecules do not diffuse intracellularly, due to their too hydrophilic character. Therefore, poly(iso-butylcyanoacrylate) (PIBCA) aqueous-cored nanocapsules have been tested as carriers to overcome the cellular delivery of AZT-TP. However, encapsulation of AZT-TP remained challenging because this molecule, due to its relatively low molecular weight, rapidly leaked out of the nanocapsules. In this study, we show that association of AZT-TP to a cationic polymer such as poly(ethyleneimine) (PEI) allowed to reach high entrapment efficiency of AZT-TP in PIBCA nanocapsules (up to 90%) as well as gradual in vitro release. The resulting hybrid PIBCA/PEI nanocapsules efficiently delivered AZT-TP in vitro to macrophages: the cellular uptake was increased by 30-fold compared to the free molecule, reaching relevant cellular concentrations for therapeutic purposes.

Nucleoside analogues, together with nucleobases and nucleotide analogues, are commonly used in the treatment of cancer and viral infections. In both cases, they act as antimetabolite agents and interfere with the synthesis of cellular or viral nucleic acids. However, the need of high doses due to the rapid elimination of these compounds, to their poor activation, and/or to their non-specific distribution, often leads to side effects and resistances. The present paper aims to review the different types of polymer nanoparticles which have been designed as drug delivery devices to address these issues. Thus, poly(alkylcyanoacrylate) nanoparticles have been demonstrated as potential carriers for antiviral nucleoside analogues, especially for anti-HIV agents, regarding both intravenous and oral routes. Nanoparticles based on polyesters such as poly(lactic acid) and poly(lactide-co-glycolide) have been used as nanocarriers for nucleosides analogues too, and especially for their ocular delivery. Albumin has shown interesting properties in the design of nanoparticles for the same application, but also for the oral administration of anticancer analogues. Finally, new hydrophilic nanoparticles consisting of cross-linked polymer network ('Nanogels') open the perspective to deliver nucleoside analogues within their active triphosphate form.

Dual-labeled phosphorothioate oligonucleotides were encapsulated in the internal aqueous core of poly(isobutylcyanoacrylate) nanocapsules with high yields. Intracellular distribution of naked and encapsulated oligomers was assessed on cultured vascular smooth muscle cells using cell fractionation and confocal microscopy methods. The results showed that cell uptake and more interestingly, nuclear accumulation of oligonucleotides were importantly enhanced when the oligonucleotides were associated to the nanocapsules as compared to the naked oligomers. These results suggested an efficient capacity of this polymeric carrier to deliver its payload to the cytosol and nucleus of smooth muscle cells.

Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction. This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20 min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles.

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controlled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.

Electroporation, using high voltage electrical pulses has been recognized as a powerful method for delivering macromolecules such as DNA and proteins in cells, or smaller molecules through the skin. Transdermal electroporation could combine targeted delivery of drugs to the skin and permeabilization of skin cells, suggesting that electroporation could be an interesting alternative for topical delivery of oligonucleotides. This work is devoted to the determination of the electroporation parameters that allow optimal delivery of oligonucleotides to the viable tissues of hairless rat skin in vitro. Phosphorothioate derivatives were preferred to the phosphodiester congeners as the former were found to be much less degraded when extracted from the tissues. Long duration (100-500 ms)--medium voltage (100-200 V)--exponentially decaying pulses appeared to be the best conditions for delivering oligonucleotides to the skin. The oligonucleotide quantity permeating the viable tissues of the skin was controlled by the selection of the electrical parameters of the pulses (voltage, pulse time and number of pulses) or by the ON concentration in the donor compartment. After delivery by electroporation, therapeutic levels of oligonucleotides were reached in the viable tissues of the skin (above 1 microM or 10 microM in intact or stripped skin respectively). Taken together, our results show that electroporation could be an interesting method for the delivery of oligonucleotides to the skin.

Antisense oligonucleotides are currently used for the specific control of the expression of a selected gene. Their putative targets are located in the cytoplasm (messenger RNA) or the nucleus (pre-messenger RNA or DNA). This approach is conditioned by the presence of the antisense molecule inside the cell at sufficient concentrations and in the appropriate compartments. We propose in this paper a simple method for the study of the cytosolic content of internalized oligonucleotides. This method is based on the selective permeabilization of the plasmic membrane by the detergent digitonin. By complexing to membrane cholesterol, the detergent creates pores through which soluble and diffusible species can escape outside the cells. The selectivity of membrane permeabilization was controlled by using compartment markers: lactate dehydrogenase (LDH) for cytosol, dextrane-rhodamine (DEX) and hexosaminidase (HAM) for endocytic vesicles and lysosomes, respectively. Optimal digitonin concentrations and incubation times have been defined to reach the following pattern of membrane permeabilization: LDH > 80%; DEX and HAM < 15%. The method was applied to monitor the quantity of extractible oligonucleotides from cells after endocytosis. The results showed that phosphodiester and phosphorothioate oligomers are readily available in the cytosol (60-50% of the internalized species), whereas those bearing a hydrophobic moiety (fluorescein, cholesterol) are less diffusible probably owing to membrane binding. Internalization and cytosol partition were found to depend on the chemical nature of the oligonucleotide, and also on the sequence and the cell type. This method could be useful for the selection of antisense molecules that exhibit the best internalization and distribution in cells, and for a more appropriate choice of control sequences in antisense studies.