Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA + RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA + RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.

The fate of cellular mRNAs was analyzed in several cell lines of lymphoid origin, after induction of apoptosis by different mechanisms. Cytoplasmic mRNAs are specifically degraded as part of the early apoptotic response. This degradation is not species restricted and is independent of the cell line, the apoptotic stimulus, the intrinsic half-life of the mRNAs, and the transcriptional status of the gene (constitutive or inducible). mRNA degradation precedes DNA fragmentation and correlates with the appearance of phosphatidylserine in the outer cell membrane. In addition, apoptosis-induced mRNA degradation is an active process that can be dissected from other apoptotic hallmarks (degradation of annexin V, DNA, and poly(ADP-ribose) polymerase [PARP]), which suggests that apoptosis-induced mRNA degradation is controlled by a distinct signaling pathway. Furthermore, mRNA degradation also occurs in vivo, specifically during thymocyte apoptosis. Taken together, these data support the notion that degradation of mRNA is a general early apoptotic event that may become a new apoptotic hallmark.Key words: RNA metabolism • caspase inhibitors • signaling pathways • in vivo apoptosis T he genetically programmed cell death process known as apoptosis is fundamental for the normal development and homeostasis of tissues and organs in multicellular organisms.The relevance of apoptosis is demonstrated both by the evolutionary conservation of basic steps in the corresponding pathways (1) and by the causal implication of aberrant apoptotic mechanisms in diseases including neurodegenerative diseases, ischemic damage, autoimmune disorders, and several forms of cancer (2-6). Apoptosis was originally characterized by morphological features such as membrane blebbing, cell shrinkage, formation of apoptotic bodies, chromatin condensation, nucleolus loss, and chromatin fragmentation into single or multiple nucleosomes (7,8). In recent years, many molecules participating in the pathways mediating apoptosis have been identified (9). Caspase activation is a feature common to most of these pathways and leads to proteolytic cleavage of specific cell substrates (10-12), including poly(ADP-ribose) polymerase (PARP), lamins, histone H1 (13), and DNA protein kinase (14,15), as well as proteins involved in cell growth, survival, and death (11). Caspases also cleave the inhibitor of caspase-dependent DNase (ICAD), thus releasing active caspase-dependent DNase (CAD), which after translocation to the nucleus fragments chromatin (16). In addition, several reports have analyzed the fate of RNA during apoptosis. Indeed, [ 3 H]uridine pulse-chase experiments detected a twofold increase in RNA degradation rates in rat thymocytes after glucocorticoid treatment (17). More recently, cleavage of both 28S ribosomal RNA (rRNA) and Ro ribonucleoprotein-associated Y RNAs during apoptosis was shown (18, 19), but nothing is known regarding the fate of cytoplasmic mRNAs.In the immune system, apoptosis plays a key role both in the control of lymphocyte maturation ...

Recent efficiency records of organic photovoltaics (OPV) highlight stability as a limiting weakness. Incorporation of stabilizers is a desirable approach for inhibiting degradationit is inexpensive and readily up-scalable. However, to date, such additives have had limited success. We show that β-carotene (BC), an inexpensive and green, naturally occurring antioxidant, dramatically improves OPV stability. When compared to nonstabilized reference devices, the accumulated power generation of PTB7:[70]­PCBM devices in the presence of BC increases by an impressive factor of 6, due to stabilization of both the burn-in and the lifetime, and by a factor of 21 for P3HT:[60]­PCBM devices, owing to a longer lifetime. Using electron spin resonance and time-resolved near-IR emission spectroscopies, we probed radical and singlet oxygen concentrations. We demonstrate that singlet oxygen sensitized by [70]­PCBM causes the “burn-in” of PTB7:[70]­PCBM devices and that BC effectively mitigates it. Our results provide an effective solution to the problem that currently limits widespread use of OPV.

Photo-initiated, oxygen-mediated degradation of the molecules in the active layer of organic photovoltaic, OPV, devices currently limits advances in the development of solar cells. To address this problem systematically and at a molecular level, it is informative to quantify the kinetics of the pertinent processes, both in solution phase and in solid films. To this end, we examined the oxygen-dependent photophysics and photochemistry of selected functionalized fullerenes, thiophene derivatives, and a subphthalocyanine commonly used in OPV devices. We find that the photosensitized production of singlet molecular oxygen, O2(a1Δg), by these molecules is a key step in the degradation process. We demonstrate that the addition of either β-carotene or astaxanthin as antioxidants can inhibit degradation by a combination of three processes: (a) deactivation of O2(a1Δg) to the oxygen ground state, O2(X3Σg−), (b) quenching of the O2(a1Δg) precursor, and (c) sacrificial reactions of the carotenoid with free radicals formed in the photo-initiated reactions. For OPV systems in which reaction with O2(a1Δg) contributes to the degradation, the first two of these processes are desired and should have appreciable impact in prolonging the longevity of OPV devices because they do not result in a chemical change of the system.

The precise mechanisms involved in the switch between the clonal expansion and contraction phases of a CD8+ T cell response remain to be fully elucidated. One of the mechanisms implicated in the contraction phase is cytokine deprivation, which triggers apoptosis in these cells. CCR2 chemokine receptor is up-regulated following IL-2 deprivation, and its ligand CCL2 plays an essential role preventing apoptosis induced by IL-2 withdrawal not only in CTLL2 cells, but also in mouse Ag-activated primary CD8+ T cells because it rescued functional CD8+ T cells from deprivation induced apoptosis, promoting proliferation in response to subsequent addition of IL-2 or to secondary antigenic challenges. Thus, up-regulation of the CCR2 upon growth factor withdrawal together with the protective effects of CCL2, represent a double-edged survival strategy, protecting cells from apoptosis and enabling them to migrate toward sites where Ag and/or growth factors are available.

The development of nonfullerene acceptors (NFAs) has led to dramatic improvements in the device efficiencies of organic photovoltaic (OPV) cells. To date it is, however, still unclear how those laboratory‐scale efficiencies transfer to commercial modules, and how stable these devices will be when processed via industrially compatible methods. Herein, the degradation behavior of lab‐scale and scalable OPV devices using similar nonfullerene‐based active layers is assessed. It is demonstrated that the scalable NFA OPV exhibits completely reversible degradation when assessed in ISOS‐O‐1 outdoor conditions, which is in contrast to the laboratory‐scale devices assessed via the indoor ISOS‐L‐2 protocol. Results from transient photovoltage (TPV) indicate the presence of charge trap formation, and a number of potential mechanisms are proposed for the selective occurrence of this in laboratory‐scale devices tested in ISOS‐L laboratory conditions—ultimately concluding that it has its origins in the different device architectures used. The study points at the risk of assessing active layer stability from laboratory‐scale devices and degradation studies alone and highlights the importance of using a diverse range of testing conditions and ISOS protocols for such assessment.

Photochemical and mechanical stability are critical in the production and application of organic solar cells. While these factors can individually be improved using different additives, there is no example of...

Degradation of perovskite solar cells (PSCs) is often found to be partially or fully reversible when the cells are allowed to recover in the dark. Unlike the dynamics of degradation, knowledge about the dynamics of PSC cell recovery is very limited. Here, we demonstrate that the PSC recovery strongly depends on the electrical bias conditions during the light-induced degradation and that it can be manipulated by applying an external electrical bias during the recovery phase. Investigation of the recovery dynamics allows us to analyze the degradation mechanisms in detail. More specifically, we aged a mixed-cation mixed-halide PSC with a n-i-p structure under illumination in open-circuit (OC) or short-circuit (SC) conditions, and periodically measured their characteristics during the recovery. PSCs aged in SC degrade faster and fully recover after the light is switched off, while the performance of the cells aged in OC does not recover but instead further decreases after the light is switched off (“drop-in-dark” effect). With the use of transient photoluminescence, secondary ion mass spectrometry, and drift-diffusion-based simulations, we hypothesize that extrinsic ion migration causes the drop-in-dark effect, by forming an electron extraction barrier at the metal oxide electron transport layer. The applied bias alleviates this effect. Our results are relevant for gaining a deeper understanding of the multiple degradation mechanisms present in perovskite solar cells, and for finding a practical way to assist their recovery.