Nelfinavir, an HIV-1 Protease Inhibitor, Induces Oxidative Stress–Mediated, Caspase-Independent Apoptosis in Leishmania Amastigotes

Visceral leishmaniasis is the most severe form of disease caused by the parasite Leishmania. It is a major concern in South America, Africa, India and the Middle East. Additionally, it has now emerged as an important opportunistic disease in patients coinfected with HIV-1. This is due, in part, to the increasing overlap between urban centers and rural areas endemic for Leishmania. Although more efficient combinatorial antiviral drug regimens for treating HIV-1 infection have been developed, the impact of such therapies on HIV-1/Leishmania coinfection is yet to be explored. In this study, we investigated the effect of nelfinavir, a well-characterized anti-HIV-1 drug, on Leishmania. Treating the parasite with nelfinavir activates events that are hallmarks of programmed cell death (also called apoptosis). Among these are oxidative stress, changes in DNA replication and fragmentation, and release of mitochondrial enzymes. Furthermore, these events occur without the participation of caspases, which are classically linked to apoptosis; however, this atypical apoptosis requires the translocation of endonuclease G from mitochondria to the cytoplasm. These findings provide insights for the design of new anti-parasitic therapies, particularly in the case of Leishmania/HIV-1 coinfections

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

GSH1 overexpression suppresses NFV-induced cell death in parasites.

<p>Stably transfected <i>L. infantum</i> strains carrying either the empty control vector Pspαzeoα (i.e. Li-pSP) or the GSH1-encoding vector PspαzeoαGSH1 were exposed to NFV (40 µM) for 24 h. Cell death was measured in terms of DNA degradation. Light gray bars refer to untreated parasites, whereas black bars relate to NFV-treated parasites (<i>n</i> = 3) (***, <i>P</i><0.001).</p

Loss of mitochondrial potential and translocation of EndoG in NFV-treated <i>L. donovani</i> amastigotes.

<p>(A) <i>L. donovani</i> axenic amastigotes were exposed to the diluent or NFV (40 µM) for the indicated time periods. Mitochondrial membrane potential (Δψ_m) was assessed through the use of the JC-1 Mitochondrial Membrane Potential Detection Kit. Data are expressed as mean ± SD of three independent experiments. (B) Western blot analysis of the mitochondrial and cytosolic fractions obtained from NFV-treated amastigotes at different intervals. Anti-EndoG immunoblots of cytosolic and mitochondrial fractions are shown, along with the loading controls for mitochondria (i.e. COX IV) and cytosol (i.e. DHFR-TS), respectively.</p

NFV induces an increase in the sub-G1 DNA-containing population.

<p>The DNA content degradation profiles of drug-sensitive and NFV-resistant axenic amastigotes (i.e. usually maintained under a constant drug pressure of 12.5 µM), which were either left untreated or treated with NFV (10 µM), were assessed by flow cytometry after cell permeabilisation and PI staining. DNA fragmentation was quantified by measuring the cell population in the sub-G1 DNA region indicated by M1 and G1 DNA peak indicated by M2. The data shown are representative of three independent experiments.</p

NFV induces degradation of genomic DNA isolated from <i>L. donovani</i> amastigotes.

<p>Genomic DNA was isolated from Ld 9518 amastigotes which have been treated with NFV or MTF (25 µM in both instances) for the indicated time periods and was resolved on 1% agarose gel. M corresponds to 1 Kb DNA ladder.</p

Cytosolic translocation of EndoG in NFV-treated <i>L. donovani</i> amastigotes is inhibited by addition of antioxidant.

<p>(A) <i>L. donovani</i> axenic amastigotes were exposed to NFV (40 µM) for 0 or 8 h with or without pre-treatment with NAC (20 mM). Western blot analysis of the cytosolic fractions obtained from the amastigotes was then performed using anti-EndoG (upper panel) and DHFR-TS (lower panel, as loading control). Densitometric analysis of blots from three of these experiments are shown in (B).</p