Evaluation of new cholinium-amino acids based room temperature ionic liquids (RTILs) as immobilization matrix for electrochemical biosensor development: proof-of-concept with trametes versicolor laccase

In this work, we present new cholinium-amino acids room temperature ionic liquids (ChAARTILs) that can be used as an efficient immobilization matrix for electrochemical biosensor development. The ideal immobilization strategy should be able to ensure the highest enzyme loading and a tight enzymatic immobilization, preserving its native structure and biological activity. In this regard, ChAARTILs present different side chains on the amino acids giving rise to van der Waals, π-π stacking and hydrogen bonding interactions. All these interactions can affect the nanomaterial organization onto the electrode surface. To this aim, we have evaluated the main electrochemical parameters, namely electroactive area (AEA) and the heterogeneous electron transfer rate constant (k0), showing how both cations and anions of room temperature ionic liquids (RTILs) can independently affect multi-walled carbon nanotubes (MWCNTs) organization. In particular, [Ch][Phe] showed the best performance in terms of AEA (3.432 cm2) and k0 (4.71·10−3 cm s−1) with a homogeneous distribution of MWCNTs bundles onto the electrodes and a faster electron transfer rate

Overview of the main methods used to combine proteins with nanosystems: absorption, bioconjugation, and encapsulation

The latest development of protein engineering allows the production of proteins having desired properties and large potential markets, but the clinical advances of therapeutical proteins are still limited by their fragility. Nanotechnology could provide optimal vectors able to protect from degradation therapeutical biomolecules such as proteins, enzymes or specific polypeptides. On the other hand, some proteins can be also used as active ligands to help nanoparticles loaded with chemotherapeutic or other drugs to reach particular sites in the body. The aim of this review is to provide an overall picture of the general aspects of the most successful approaches used to combine proteins with nanosystems. This combination is mainly achieved by absorption, bioconjugation and encapsulation. Interactions of nanoparticles with biomolecules and caveats related to protein denaturation are also pointed out. A clear understanding of nanoparticle-protein interactions could make possible the design of precise and versatile hybrid nanosystems. This could further allow control of their pharmacokinetics as well as activity, and safety

Intravitreal Gene Therapy vs. Natural History in Patients With Leber Hereditary Optic Neuropathy Carrying the m.11778G>A <i>ND4</i> Mutation: Systematic Review and Indirect Comparison.

Objective: This work aimed to compare the evolution of visual outcomes in Leber hereditary optic neuropathy (LHON) patients treated with intravitreal gene therapy to the spontaneous evolution in prior natural history (NH) studies. Design: A combined analysis of two phase three randomized, double-masked, sham-controlled studies (REVERSE and RESCUE) and their joint long-term extension trial (CLIN06) evaluated the efficacy of rAAV2/2-ND4 vs. 11 pooled NH studies used as an external control. Subjects: The LHON subjects carried the m.11778G>A ND4 mutation and were aged ≥15 years at onset of vision loss. Methods: A total of 76 subjects received a single intravitreal rAAV2/2-ND4 injection in one eye and sham injection in the fellow eye within 1 year after vision loss in REVERSE and RESCUE. Both eyes were considered as treated due to the rAAV2/2-ND4 treatment efficacy observed in the contralateral eyes. Best corrected visual acuity (BCVA) from REVERSE, RESCUE, and CLIN06 up to 4.3 years after vision loss was compared to the visual acuity of 208 NH subjects matched for age and ND4 genotype. The NH subjects were from a LHON registry (REALITY) and from 10 NH studies. A locally estimated scatterplot smoothing (LOESS), non-parametric, local regression model was used to modelize visual acuity curves over time, and linear mixed model was used for statistical inferences. Main Outcome Measures: The main outcome measure was evolution of visual acuity from 12 months after vision loss, when REVERSE and RESCUE patients had been treated with rAAV2/2-ND4. Results: The LOESS curves showed that the BCVA of the treated patients progressively improved from month 12 to 52 after vision loss. At month 48, there was a statistically and clinically relevant difference in visual acuity of -0.33 logarithm of the minimal angle of resolution (LogMAR) (16.5 ETDRS letters equivalent) in favor of treated eyes vs. NH eyes (p p p Conclusions: The m.11778G>A LHON patients treated with rAAV2/2-ND4 exhibited an improvement of visual acuity over more than 4 years after vision loss to a degree not demonstrated in NH studies. Clinical Trial Registration: NCT02652767, NCT02652780, NCT03406104, and NCT03295071

Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy

Steps toward determination of the size and structure of the broad-line region in active galactic nuclei. 5: Variability of the ultraviolet continuum and emission lines of NGC 3783

We report on the results of intensive ultraviolet spectral monitoring of the Seyfert 1 galaxy NGC 3783. The nucleus of NGC 3783 was observed with the International Ultraviolet Explorer satellite on a regular basis for a total of 7 months, once every 4 days for the first 172 days and once every other day for the final 50 days. Significant variability was observed in both continuum and emission-line fluxes. The light curves for the continuum fluxes exhibited two well-defined local minima or 'dips,' the first lasting is less than or approximately 20 days and the second is less than or approximately 4 days, with additional episodes of relatively rapid flickering of approximately the same amplitude. As in the case of NGC 5548 (the only other Seyfert galaxy that has been the subject of such an intensive, sustained monitoring effort), the largest continuum variations were seen at the shortest wavelengths, so that the continuum became 'harder' when brighter. The variations in the continuum occurred simultaneously at all wavelengths (delta(t) is less than 2 days). Generally, the amplitude of variability of the emission lines was lower than (or comparable to) that of the continuum. Apart from Mg II (which varied little) and N V (which is relatively weak and badly blended with Ly(alpha), the light curves of the emission lines are very similar to the continuum light curves, in each case with a small systematic delay or 'lag.' As for NGC 5548, the highest ionization lines seem to respond with shorter lags than the lower ionization lines. The lags found for NGC 3783 are considerably shorter than those obtained for NGC 5548, with values of (formally) approximately 0 days for He II + O III), and approximately 4 days for Ly(alpha) and C IV. The data further suggest lags of approximately 4 days for Si IV + O IV) and 8-30 days for Si III + C III). Mg II lagged the 1460 A continuum by approximately 9 days, although this result depends on the method of measuring the line flux and may in fact be due to variability of the underlying Fe II lines. Correlation analysis further shows that the power density spectrum contains substantial unresolved power over timescales of is less than or approximately 2 days, and that the character of the continuum variability may change with time

Indirect Comparison of Lenadogene Nolparvovec Gene Therapy Versus Natural History in Patients with Leber Hereditary Optic Neuropathy Carrying the m.11778G\u3eA MT-ND4 Mutation

INTRODUCTION: Lenadogene nolparvovec is a promising novel gene therapy for patients with Leber hereditary optic neuropathy (LHON) carrying the m.11778G\u3eA ND4 mutation (MT-ND4). A previous pooled analysis of phase 3 studies showed an improvement in visual acuity of patients injected with lenadogene nolparvovec compared to natural history. Here, we report updated results by incorporating data from the latest phase 3 trial REFLECT in the pool, increasing the number of treated patients from 76 to 174. METHODS: The visual acuity of 174 MT-ND4-carrying patients with LHON injected in one or both eyes with lenadogene nolparvovec from four pooled phase 3 studies (REVERSE, RESCUE and their long-term extension trial RESTORE; and REFLECT trial) was compared to the spontaneous evolution of an external control group of 208 matched patients from 11 natural history studies. RESULTS: Treated patients showed a clinically relevant and sustained improvement in their visual acuity when compared to natural history. Mean improvement versus natural history was - 0.30 logMAR (+ 15 ETDRS letters equivalent) at last observation (P \u3c 0.01) with a maximal follow-up of 3.9 years after injection. Most treated eyes were on-chart as compared to less than half of natural history eyes at 48 months after vision loss (89.6% versus 48.1%; P \u3c 0.01) and at last observation (76.1% versus 44.4%; P \u3c 0.01). When we adjusted for covariates of interest (gender, age of onset, ethnicity, and duration of follow-up), the estimated mean gain was - 0.43 logMAR (+ 21.5 ETDRS letters equivalent) versus natural history at last observation (P \u3c 0.0001). Treatment effect was consistent across all phase 3 clinical trials. Analyses from REFLECT suggest a larger treatment effect in patients receiving bilateral injection compared to unilateral injection. CONCLUSION: The efficacy of lenadogene nolparvovec in improving visual acuity in MT-ND4 LHON was confirmed in a large cohort of patients, compared to the spontaneous natural history decline. Bilateral injection of gene therapy may offer added benefits over unilateral injection. TRIAL REGISTRATION NUMBERS: NCT02652780 (REVERSE); NCT02652767 (RESCUE); NCT03406104 (RESTORE); NCT03293524 (REFLECT); NCT03295071 (REALITY)

The whole earth blazar telescope campaign on the intermediate BL Lac object 3C 66A in 2007-2008

Prompted by a high optical state in 2007 September, the Whole Earth Blazar Telescope consortium organized an intensive optical, near-IR (JHK) and radio observing campaign on the intermediate BL Lac object 3C 66A throughout the fall and winter of 2007-2008. In this paper, we present data from 28 observatories in 12 countries, covering the observing season from late 2007 July through 2008 February. The source remained in a high optical state throughout the observing period and exhibited several bright flares on timescales of 10 days. This included an exceptional outburst around 2007 September 15-20, reaching a peak brightness at R 13.4. Our campaign revealed microvariability with flux changes up to |dR/dt| 0.02 mag hr-1. Our observations do not reveal evidence for systematic spectral variability in the overall high state covered by our campaign, in agreement with previous results. In particular, we do not find evidence for spectral hysteresis in 3C 66A for which hints were found in an earlier campaign in a somewhat lower flux state. We also did not find any evidence for spectral lags in the discrete correlation functions between different optical bands. We infer a value of the magnetic field in the emission region of B 19 e 2/7B τ-6/7h D 13/71 G, where eB is the magnetic field equipartition fraction, τh is the shortest observed variability timescale in units of hours, and D 1 is the Doppler factor in units of 10. From the lack of systematic spectral variability, we can derive an upper limit on the Doppler factor, D ≤ 28 τ-1/8h e 3/16B. This is in perfect agreement with superluminal motion measurements with the VLBI/VLBA of βapp ≤ 27 and argues against models with very high Lorentz factors of Γ ≳ 50, required for a one-zone synchrotron-self-Compton interpretation of some high-frequency-peaked BL Lac objects detected at TeV γ-ray energies. © 2009 The American Astronomical Society