Cadmium (Cd) is a non‐essential heavy metal that may be toxic or even lethal to plants as it can be easily taken up by the roots and loaded into the xylem to the leaves. Using soybean roots (Glycine ...max L.) DM 4800, we have analysed various parameters related to reactive oxygen metabolism and nitric oxide (NO) during a 6 day Cd exposure. A rise in H₂O₂ and NO, and to a lesser extent O₂ ·⁻ content was observed after 6 h exposure with a concomitant increase in lipid peroxidation and carbonyl group content. Both oxidative markers were significantly reduced after 24 h. A second, higher wave of O₂ ·⁻ production was also observed after 72 h of exposure followed by a reduction until the end of the treatment. NOX and glicolate oxidase activity might be involved in the initial Cd‐induced reactive oxygen species (ROS) production and it appears that other sources may also participate. The analysis of antioxidative enzymes showed an increase in glutathione‐S‐transferase activity and in transcript levels and activity of enzymes involved in the ascorbate‐glutathione cycle and the NADPH‐generating enzymes. These results suggest that soybean is able to respond rapidly to oxidative stress imposed by Cd by improving the availability of NADPH necessary for the ascorbate‐glutathione cycle.
Cadmium (Cd) toxicity has been widely studied in different plant species; however, the mechanism involved in its toxicity as well as the cell response against the metal have not been well ...established. In this work, using pea (Pisum sativum) plants, we studied the effect of Cd on antioxidants, reactive oxygen species (ROS), and nitric oxide (NO) metabolism of leaves using different cellular, molecular, and biochemical approaches. The growth of pea plants with 50 μM CdCl₂ affected differentially the expression of superoxide dismutase (SOD) isozymes at both transcriptional and posttranscriptional levels, giving rise to a SOD activity reduction. The copper/zinc-SOD down-regulation was apparently due to the calcium (Ca) deficiency induced by the heavy metal. In these circumstances, the overproduction of the ROS hydrogen peroxide and superoxide could be observed in vivo by confocal laser microscopy, mainly associated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals was prevented by exogenous Ca. On the other hand, the NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO.
ABSTRACT
Growth of pea (Pisum sativum L.) plants with 50 µm CdCl2 for 15 d produced a reduction in the number and length of lateral roots, and changes in structure of the principal roots affecting ...the xylem vessels. Cadmium induced a reduction in glutathione (GSH) and ascorbate (ASC) contents, and catalase (CAT), GSH reductase (GR) and guaiacol peroxidase (GPX) activities. CuZn‐superoxide dismutase (SOD) activity was also diminished by the Cd treatment, although Mn‐SOD was slightly increased. CAT and CuZn‐SOD were down‐regulated at transcriptional level, while Mn‐SOD, Fe‐SOD and GR were up‐regulated. Analysis of reactive oxygen species (ROS) and nitric oxide (NO) levels by fluorescence and confocal laser microscopy (CLM) showed an over‐accumulation of O2.− and H2O2, and a reduction in the NO content in lateral and principal roots. ROS overproduction was dependent on changes in intracellular Ca+2 content, and peroxidases and NADPH oxidases were involved. Cadmium also produced an increase in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) contents. The rise of ET and ROS, and the NO decrease are in accordance with senescence processes induced by Cd, and the increase of JA and SA could regulate the cellular response to cope with damages imposed by cadmium.
Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. ...Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.
Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted. Although it is generally ...believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels, suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs. Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.
The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are ...limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay).
Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR.
vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs.
We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.
ABSTRACT
In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D) (23 mm) applied by foliar spraying ...was investigated. The concentration of 2,4‐D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl‐CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over‐accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4‐D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.
In this work, it has been evidenced that reactive oxygen species (ROS) overproduction is a key point in the effect of high concentrations of 2,4‐D, being peroxisomal fatty acid β‐oxidation one of the main sources, in addition to xanthine oxidase. ROS have a differentiated role in young and adult tissue, being involved in epinasty in young leaves, and senescence in old leaves. Ethylene can also be involved in epinasty and senescence and could contribute to ROS production. The role of ROS in epinasty is related with cell wall expansion, vascular tissue proliferation and oxidative processes which can affect the cytoskeleton giving rise to the curly of leaves. The oxidative burst caused by 2,4‐D leads to the activation of some stress and defence proteins such as PRP4‐A and HSP 71.1, which could contribute to mitigate damages induced by the herbicide.
Exogenous NO prevents oxidative damage in response to heavy metal stress, alleviating plant fitness loss, while endogenous NO should be closely regulated, and NO-dependent signalling pathways are ...involved in plant resistance.
Abstract
Anthropogenic activities, such as industrial processes, mining, and agriculture, lead to an increase in heavy metal concentrations in soil, water, and air. Given their stability in the environment, heavy metals are difficult to eliminate and can constitute a human health risk by entering the food chain through uptake by crop plants. An excess of heavy metals is toxic for plants, which have various mechanisms to prevent their accumulation. However, once metals enter the plant, oxidative damage sometimes occurs, which can lead to plant death. Initial production of nitric oxide (NO), which may play a role in plant perception, signalling, and stress acclimation, has been shown to protect against heavy metals. Very little is known about NO-dependent mechanisms downstream from signalling pathways in plant responses to heavy metal stress. In this review, using bioinformatic techniques, we analyse studies of the involvement of NO in plant responses to heavy metal stress, its possible role as a cytoprotective molecule, and its relationship with reactive oxygen species. Some conclusions are drawn and future research perspectives are outlined to further elucidate the signalling mechanisms underlying the role of NO in plant responses to heavy metal stress.
Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. ...As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4
T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic (
= 18) and antiretroviral therapy (ART)-treated subjects (
= 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency.
Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.
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