Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways

Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Large-scale unit commitment under uncertainty: an updated literature survey

The Unit Commitment problem in energy management aims at finding the optimal production schedule of a set of generation units, while meeting various system-wide constraints. It has always been a large-scale, non-convex, difficult problem, especially in view of the fact that, due to operational requirements, it has to be solved in an unreasonably small time for its size. Recently, growing renewable energy shares have strongly increased the level of uncertainty in the system, making the (ideal) Unit Commitment model a large-scale, non-convex and uncertain (stochastic, robust, chance-constrained) program. We provide a survey of the literature on methods for the Uncertain Unit Commitment problem, in all its variants. We start with a review of the main contributions on solution methods for the deterministic versions of the problem, focussing on those based on mathematical programming techniques that are more relevant for the uncertain versions of the problem. We then present and categorize the approaches to the latter, while providing entry points to the relevant literature on optimization under uncertainty. This is an updated version of the paper "Large-scale Unit Commitment under uncertainty: a literature survey" that appeared in 4OR 13(2), 115--171 (2015); this version has over 170 more citations, most of which appeared in the last three years, proving how fast the literature on uncertain Unit Commitment evolves, and therefore the interest in this subject

From the Surface Ocean to the Seafloor: Linking Modern and Paleo-Genetics at the Sabrina Coast, East Antarctica (IN2017_V01)

With ongoing climate change, research into the biological changes occurring in particularly vulnerable ecosystems, such as Antarctica, is critical. The Totten Glacier region, Sabrina Coast, is currently experiencing some of the highest rates of thinning across all East Antarctica. An assessment of the microscopic organisms supporting the ecosystem of the marginal sea-ice zone over the continental rise is important, yet there is a lack of knowledge about the diversity and distribution of these organisms throughout the water column, and their occurrence and/or preservation in the underlying sediments. Here, we provide a taxonomic overview of the modern and ancient marine bacterial and eukaryotic communities of the Totten Glacier region, using a combination of 16S and 18S rRNA amplicon sequencing (modern DNA) and shotgun metagenomics (sedimentary ancient DNA, sedaDNA). Our data show considerable differences between eukaryote and bacterial signals in the water column versus the sediments. Proteobacteria and diatoms dominate the bacterial and eukaryote composition in the upper water column, while diatoms, dinoflagellates, and haptophytes notably decrease in relative abundance with increasing water depth. Little diatom sedaDNA is preserved in the sediments, which are instead dominated by Proteobacteria and Retaria. We compare the diatom microfossil and sedaDNA record and link the weak preservation of diatom sedaDNA to DNA degradation while sinking through the water column to the seafloor. This study provides the first assessment of DNA transfer from ocean waters to sediments and an overview of the microscopic communities occurring in the climatically important Totten Glacier region

Beyond Implications and Applications: the Story of ‘Safety by Design’

Using long-term anthropological observations at the Center for Biological and Environmental Nanotechnology in Houston, Texas, the article demonstrates in detail the creation of new objects, new venues and new modes of veridiction which have reoriented the disciplines of materials chemistry and nanotoxicology. Beginning with the confusion surrounding the meaning of ‘implications’ and ‘applications’ the article explores the creation of new venues (CBEN and its offshoot the International Council on Nanotechnology); it then demonstrates how the demands for a responsible, safe or ethical science were translated into new research and experiment in and through these venues. Finally it shows how ‘safety by design’ emerged as a way to go beyond implications and applications, even as it introduced a whole new array of controversies concerning its viability, validity and legitimacy

Deregulated expression of hnRNP A/B proteins in human non-small cell lung cancer: parallel assessment of protein and mRNA levels in paired tumour/non-tumour tissues

<p>Abstract</p> <p>Background</p> <p>Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type (hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC) to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2.</p> <p>Methods</p> <p>We combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient.</p> <p>Results</p> <p>Our study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%), followed by A3 (52%) and A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1, suggesting that different mechanisms underlie their deregulation.</p> <p>Conclusion</p> <p>Our results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.</p

Specific Responses of Salmonella enterica to Tomato Varieties and Fruit Ripeness Identified by In Vivo Expression Technology

Recent outbreaks of vegetable-associated gastroenteritis suggest that enteric pathogens colonize, multiply and persist in plants for extended periods of time, eventually infecting people. Genetic and physiological pathways, by which enterics colonize plants, are still poorly understood.To better understand interactions between Salmonella enterica sv. Typhimurium and tomatoes, a gfp-tagged Salmonella promoter library was screened inside red ripe fruits. Fifty-one unique constructs that were potentially differentially regulated in tomato relative to in vitro growth were identified. The expression of a subset of these promoters was tested in planta using recombinase-based in vivo expression technology (RIVET) and fitness of the corresponding mutants was tested. Gene expression in Salmonella was affected by fruit maturity and tomato cultivar. A putative fadH promoter was upregulated most strongly in immature tomatoes. Expression of the fadH construct depended on the presence of linoleic acid, which is consistent with the reduced accumulation of this compound in mature tomato fruits. The cysB construct was activated in the fruit of cv. Hawaii 7997 (resistant to a race of Ralstonia solanacearum) more strongly than in the universally susceptible tomato cv. Bonny Best. Known Salmonella motility and animal virulence genes (hilA, flhDC, fliF and those encoded on the pSLT virulence plasmid) did not contribute significantly to fitness of the bacteria inside tomatoes, even though deletions of sirA and motA modestly increased fitness of Salmonella inside tomatoes.This study reveals the genetic basis of the interactions of Salmonella with plant hosts. Salmonella relies on a distinct set of metabolic and regulatory genes, which are differentially regulated in planta in response to host genotype and fruit maturity. This enteric pathogen colonizes tissues of tomatoes differently than plant pathogens, and relies little on its animal virulence genes for persistence within the fruit