Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action

BACKGROUND: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. CONCLUSIONS/SIGNIFICANCE: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors

Targeting deficiencies in the TLR5 mediated vaginal response to treat female recurrent urinary tract infection

Abstract The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle

Mutations in Wnt2 Alter Presynaptic Motor Neuron Morphology and Presynaptic Protein Localization at the Drosophila Neuromuscular Junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins

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

Localization and broadband follow-up of the gravitational-wave transient GW150914

A gravitational-wave (GW) transient was identified in data recorded by the Advanced Laser Interferometer Gravitational-wave Observatory (LIGO) detectors on 2015 September 14. The event, initially designated G184098 and later given the name GW150914, is described in detail elsewhere. By prior arrangement, preliminary estimates of the time, significance, and sky location of the event were shared with 63 teams of observers covering radio, optical, near-infrared, X-ray, and gamma-ray wavelengths with ground- and space-based facilities. In this Letter we describe the low-latency analysis of the GW data and present the sky localization of the first observed compact binary merger. We summarize the follow-up observations reported by 25 teams via private Gamma-ray Coordinates Network circulars, giving an overview of the participating facilities, the GW sky localization coverage, the timeline, and depth of the observations. As this event turned out to be a binary black hole merger, there is little expectation of a detectable electromagnetic (EM) signature. Nevertheless, this first broadband campaign to search for a counterpart of an Advanced LIGO source represents a milestone and highlights the broad capabilities of the transient astronomy community and the observing strategies that have been developed to pursue neutron star binary merger events. Detailed investigations of the EM data and results of the EM follow-up campaign are being disseminated in papers by the individual teams

Search for gravitational waves from Scorpius X-1 in the second Advanced LIGO observing run with an improved hidden Markov model

We present results from a semicoherent search for continuous gravitational waves from the low-mass x-ray binary Scorpius X-1, using a hidden Markov model (HMM) to track spin wandering. This search improves on previous HMM-based searches of LIGO data by using an improved frequency domain matched filter, the J-statistic, and by analyzing data from Advanced LIGO's second observing run. In the frequency range searched, from 60 to 650 Hz, we find no evidence of gravitational radiation. At 194.6 Hz, the most sensitive search frequency, we report an upper limit on gravitational wave strain (at 95% confidence) of h095%=3.47×10-25 when marginalizing over source inclination angle. This is the most sensitive search for Scorpius X-1, to date, that is specifically designed to be robust in the presence of spin wandering. © 2019 American Physical Society

Search for gravitational waves from Scorpius X-1 in the second Advanced LIGO observing run with an improved hidden Markov model

We present results from a semicoherent search for continuous gravitational waves from the low-mass x-ray binary Scorpius X-1, using a hidden Markov model (HMM) to track spin wandering. This search improves on previous HMM-based searches of LIGO data by using an improved frequency domain matched filter, the J-statistic, and by analyzing data from Advanced LIGO’s second observing run. In the frequency range searched, from 60 to 650 Hz, we find no evidence of gravitational radiation. At 194.6 Hz, the most sensitive search frequency, we report an upper limit on gravitational wave strain (at 95% confidence) of h95%0=3.47×10−25 when marginalizing over source inclination angle. This is the most sensitive search for Scorpius X-1, to date, that is specifically designed to be robust in the presence of spin wandering

Gravitational Waves and Gamma-Rays from a Binary Neutron Star Merger: GW170817 and GRB 170817A

On 2017 August 17, the gravitational-wave event GW170817 was observed by the Advanced LIGO and Virgo detectors, and the gamma-ray burst (GRB) GRB 170817A was observed independently by the Fermi Gamma-ray Burst Monitor, and the Anti-Coincidence Shield for the Spectrometer for the International Gamma-Ray Astrophysics Laboratory. The probability of the near-simultaneous temporal and spatial observation of GRB 170817A and GW170817 occurring by chance is $5.0\times {10}^{-8}$. We therefore confirm binary neutron star mergers as a progenitor of short GRBs. The association of GW170817 and GRB 170817A provides new insight into fundamental physics and the origin of short GRBs. We use the observed time delay of $(+1.74\pm 0.05)\,{\rm{s}}$ between GRB 170817A and GW170817 to: (i) constrain the difference between the speed of gravity and the speed of light to be between $-3\times {10}^{-15}$ and $+7\times {10}^{-16}$ times the speed of light, (ii) place new bounds on the violation of Lorentz invariance, (iii) present a new test of the equivalence principle by constraining the Shapiro delay between gravitational and electromagnetic radiation. We also use the time delay to constrain the size and bulk Lorentz factor of the region emitting the gamma-rays. GRB 170817A is the closest short GRB with a known distance, but is between 2 and 6 orders of magnitude less energetic than other bursts with measured redshift. A new generation of gamma-ray detectors, and subthreshold searches in existing detectors, will be essential to detect similar short bursts at greater distances. Finally, we predict a joint detection rate for the Fermi Gamma-ray Burst Monitor and the Advanced LIGO and Virgo detectors of 0.1-1.4 per year during the 2018-2019 observing run and 0.3-1.7 per year at design sensitivity

All-sky search for gravitational-wave bursts in the second joint LIGO-Virgo run

We present results from a search for gravitational-wave bursts in the data collected by the LIGO and Virgo detectors between July 7, 2009 and October 20, 2010: data are analyzed when at least two of the three LIGO-Virgo detectors are in coincident operation, with a total observation time of 207 days. The analysis searches for transients of duration < 1 s over the frequency band 64-5000 Hz, without other assumptions on the signal waveform, polarization, direction or occurrence time. All identified events are consistent with the expected accidental background. We set frequentist upper limits on the rate of gravitational-wave bursts by combining this search with the previous LIGO-Virgo search on the data collected between November 2005 and October 2007. The upper limit on the rate of strong gravitational-wave bursts at the Earth is 1.3 events per year at 90% confidence. We also present upper limits on source rate density per year and Mpc^3 for sample populations of standard-candle sources. As in the previous joint run, typical sensitivities of the search in terms of the root-sum-squared strain amplitude for these waveforms lie in the range 5 10^-22 Hz^-1/2 to 1 10^-20 Hz^-1/2. The combination of the two joint runs entails the most sensitive all-sky search for generic gravitational-wave bursts and synthesizes the results achieved by the initial generation of interferometric detectors.Comment: 15 pages, 7 figures: data for plots and archived public version at https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=70814&version=19, see also the public announcement at http://www.ligo.org/science/Publication-S6BurstAllSky