Excited-state dynamics of [Ru(S–Sbpy)(bpy)2]2+to form long-lived localized triplet states

The novel photosensitizer [Ru( S−S bpy)(bpy) 2 ] 2+ harbors two distinct sets of excited states in the UV/Vis region of the absorption spectrum located on either bpy or S−S bpy ligands. Here, we address the question of whether following excitation into these two types of states could lead to the formation of different long-lived excited states from where energy transfer to a reactive species could occur. Femtosecond transient absorption spectros- copy identifies the formation of the final state within 80 fs for both excitation wavelengths. The recorded spectra hint at very similar dynamics following excitation toward either the parent or sulfur- decorated bpy ligands, indicating ultrafast interconversion into a unique excited-state species regardless of the initial state. Non-adiabatic surface hopping dynamics simulations show that ultrafast spin−orbit-mediated mixing of the states within less than 50 fs strongly increases the localization of the excited electron at the S−S bpy ligand. Extensive structural relaxation within this sulfurated ligand is possible, via S−S bond cleavage that results in triplet state energies that are lower than those in the analogue [Ru(bpy)3 ]2+ . This structural relaxation upon localization of the charge on S−S bpy is found to be the reason for the formation of a single long-lived species independent of the excitation wavelength

New World Hantaviruses Activate IFNλ Production in Type I IFN-Deficient Vero E6 Cells

Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs.Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus propagation

Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention

Scheme displaying the emergence of swa-resistant and swa-dependent RML prions, and their transmission through various cell lines.

<p>Initially, PK1 or PK1-derived cells were infected with RML prions, cultured in the presence of swa, and prions secreted into the conditioned medium (CM) were concentrated (CCM) and used to infect fresh batches of cells, also in the constant presence of swa. This cycle was repeated at least once more. The resulting prions were further propagated under various conditions. Large circles indicate cell lines; small circles indicate prions; horizontal arrows represent propagation of infected cells and vertical arrows transfer of prions; <#> indicates a prion sample whose swa resistance is reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-t001" target="_blank">Table 1</a> and/or in one of the further figures. <b>A.</b> CCM from PK1 cells (large green disks), collected after the first infection (<1>) in the presence of swa (red arrows), contained low titers of swa-sensitive prions (small blue disks). After transfer to a fresh batch of PK1 cells in the presence of swa, infectivity dropped below detectability (). <b>B.</b> AMO10 cells (large yellow disks) infected with RML in the presence of swa yielded swa-resistant prions (small red disks) (<2>); upon further transmission to AMO10 cells (large yellow disks) in the presence of swa the prions remained swa resistant; upon transmission to PK1 cells (large green disks) in the presence of swa the prions developed swa dependence. When transferred in the absence of swa the prions became swa sensitive (small, blue disks), semi-resistant (small orange disks) or swa dependent (small black disks) depending on whether they had previously been cultured with (red arrows) or without swa (blue arrows), and on the cell line (CAD cells violet disks). <b>C.</b> In the case of 2E4 cells, swa-dependent prions (small black disks) were recovered after the first transfer of CCM in the presence of swa (<3>); these prions remained swa dependent regardless of culture conditions or cell line.</p

Standard Scrapie Cell Assay of swa-resistant and swa-dependent RML prions transferred to PK1 cells and propagated in the presence or absence of swa.

<p>AMO10 and 2E4 cells were infected with RML prions, cultured in the presence of swa, and prions secreted into the conditioned medium (CM) were concentrated (CCM) and used to infect fresh batches of cells in the constant presence of swa. This cycle was repeated once more, whereupon the prions were transferred to PK1 cells in the presence or absence of swa (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g002" target="_blank">Figure 2B, C</a>). CCM was analyzed by the SSCA on PK1 cells in the absence (blue line) or presence (red, dashed line) of swa. <b>A.</b> Swa-resistant AMO10-derived prions developed swa dependence when propagated in PK1 cells in the presence of swa (<4>); when propagated in PK1 cells in the absence of swa, they reverted to swa sensitivity (<5>). In contrast, swa-dependent 2E4-derived prions remained swa dependent in PK1 cells, whether they were propagated in the presence (<7>) or absence (<8>) of swa. RIs are the reciprocals of the dilutions yielding 500 PrP^res positive cells per 20000 cells. Q_swa = RI_cell/RI_cell+swa reflects inhibition by swa and may be compared to the value for swa-sensitive, brain-derived RML prions. <b>B.</b> AMO10- or 2E4-derived prions that had acquired or retained swa dependence after propagation in PK1 cells in the presence of swa were either continuously propagated in the presence of swa (<10>, <14>) or cultured for eleven splits in the absence of swa (<11>, <15>). Once swa dependent, they remained dependent, whether propagated in the presence or absence of swa. RIs are the reciprocals of the dilutions required to yield 1000 PrP^res positive cells per 20000 cells. Q_swa for RML^brain is unusually high in this assay and that of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g004" target="_blank">Figure 4C</a>, leading to the low Q_rel values shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-t001" target="_blank">Table 1</a>.</p

Acquisition of Drug Resistance and Dependence by Prions

<div><p>We have reported that properties of prion strains may change when propagated in different environments. For example, when swainsonine-sensitive 22L prions were propagated in PK1 cells in the presence of swainsonine, drug-resistant variants emerged. We proposed that prions constitute quasi- populations comprising a range of variants with different properties, from which the fittest are selected in a particular environment. Prion populations developed heterogeneity even after biological cloning, indicating that during propagation mutation-like processes occur at the conformational level. Because brain-derived 22L prions are naturally swainsonine resistant, it was not too surprising that prions which had become swa sensitive after propagation in cells could revert to drug resistance. Because RML prions, both after propagation in brain or in PK1 cells, are swainsonine sensitive, we investigated whether it was nonetheless possible to select swainsonine-resistant variants by propagation in the presence of the drug. Interestingly, this was not possible with the standard line of PK1 cells, but in certain PK1 sublines not only swainsonine-resistant, but even swainsonine-dependent populations (i.e. that propagated more rapidly in the presence of the drug) could be isolated. Once established, they could be passaged indefinitely in PK1 cells, even in the absence of the drug, without losing swainsonine dependence. The misfolded prion protein (PrP^Sc) associated with a swainsonine-dependent variant was less rapidly cleared in PK1 cells than that associated with its drug-sensitive counterpart, indicating that likely structural differences of the misfolded PrP underlie the properties of the prions. In summary, propagation of prions in the presence of an inhibitory drug may not only cause the selection of drug-resistant prions but even of stable variants that propagate more efficiently in the presence of the drug. These adaptations are most likely due to conformational changes of the abnormal prion protein.</p> </div

Standard Scrapie Cell Assay of swa-resistant and swa-dependent RML prions transferred to CAD cells in the absence of swa.

<p>Swa-resistant AMO10-derived prions and swa-dependent 2E4-derived prions were transferred to CAD cells and propagated in the absence of swa for 8 splits. Concentrated conditioned medium from these cultures was then analyzed by the SSCA on PK1 cells in the absence (blue line) or presence (red, dashed line) of swa. RIs are the reciprocals of the dilutions yielding 1000 PrP^res-positive cells per 20000 cells. Q_swa = RI_cell/RI_cell+swa reflects inhibition by swa and may be compared with the value for swa-sensitive, brain-derived RML prions (rightmost panel). Swa-resistant AMO10-derived prions reverted to swa sensitivity (<18>), while swa-dependent 2E4-derived prions remained dependent (<19>).</p

Relative swa resistance of various prion populations.

a<p>Prion samples were assayed by the Standard Scrapie Cell Assay (SSCA) on PK1 cells in the absence or presence of swainsonine (swa, 1 µg/ml).</p>b<p>as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g002" target="_blank">Figure 2</a> (cell-derived samples) or <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g008" target="_blank">Figure 8</a> (brain-derived samples).</p>c<p>Q_rel = Q_sample/Q_RML where Q = RI_PK1/RI_PK1+swa and RI_cell = reciprocal of the dilution required to yield specified designated number of PrP^res positive cells per 20000 cells.</p><p>nid = no infectivity detected; nrd = no ratio determinable.</p>*<p>Q_RML was exceptionally high in the experiments of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g004" target="_blank">Figures 4C</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g005" target="_blank">5B</a> (about 2.7 rather than 1.5–2 logs), resulting in what may be misleadingly low values for Q_rel in the associated samples.</p

Standard Scrapie Cell Assay of swa-resistant and swa-dependent RML prions propagated in the presence or absence of swa.

<p>AMO10 and 2E4 cells were infected with RML prions, cultured in the presence of swa, and prions secreted into the conditioned medium (CM) were concentrated (CCM) and used to infect fresh batches of cells, also in the constant presence of swa. This cycle was repeated once more; the infected cells were cultured in the presence of swa for an extended period of time and then divided into two batches, of which one continued to be propagated in the presence of swa for nine splits while the other was propagated in parallel in the absence of swa (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g002" target="_blank">Figure 2B, C</a>). CCM from these cultures was then analyzed by the SSCA on PK1 cells in the absence (blue line) or presence (red, dashed line) of swa. RIs are the reciprocals of the dilutions required to yield 1000 PrP^res positive cells per 20000 cells. Q_swa = RI_cell/RI_cell+swa indicates the inhibitory effect of swa. <b>A.</b> Swa-resistant AMO10-derived prions that were constantly propagated in the presence of swa (<13>) remained swa resistant, but reverted to semi-resistance when propagated for nine splits in the absence of swa (<12>). <b>B.</b> Swa-dependent 2E4-derived prions remained swa dependent whether propagated for 9 splits in the presence of swa (<16>) or in its absence (<17>). <b>C.</b> Swa-sensitive, brain-derived RML prions. Q_swa for RML^brain was unusually high in this assay and that of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-g005" target="_blank">Figure 5B</a>, leading to the low Q_rel values shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003158#ppat-1003158-t001" target="_blank">Table 1</a>.</p