Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.     

Genetic and Structural Analysis of SARS-CoV-2 Spike Protein for Universal Epitope Selection

Evaluation of immunogenic epitopes for universal vaccine development in the face of ongoing SARS-CoV-2 evolution remains a challenge. Herein, we investigate the genetic and structural conservation of an immunogenically relevant epitope (C662–C671) of spike (S) protein across SARS-CoV-2 variants to determine its potential utility as a broad-spectrum vaccine candidate against coronavirus diseases. Comparative sequence analysis, structural assessment, and molecular dynamics simulations of C662–C671 epitope were performed. Mathematical tools were employed to determine its mutational cost. We found that the amino acid sequence of C662–C671 epitope is entirely conserved across the observed major variants of SARS-CoV-2 in addition to SARS-CoV. Its conformation and accessibility are predicted to be conserved, even in the highly mutated Omicron variant. Costly mutational rate in the context of energy expenditure in genome replication and translation can explain this strict conservation. These observations may herald an approach to developing vaccine candidates for universal protection against emergent variants of coronavirus.     

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.     

A common neurobiology for pain and pleasure

Pain and pleasure are powerful motivators of behaviour and have historically been considered opposites. Emerging evidence from the pain and reward research fields points to extensive similarities in the anatomical substrates of painful and pleasant sensations. Recent molecular-imaging and animal studies have demonstrated the important role of the opioid and dopamine systems in modulating both pain and pleasure. Understanding the mutually inhibitory effects that pain and reward processing have on each other, and the neural mechanisms that underpin such modulation, is important for alleviating unnecessary suffering and improving well-being.     

Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats

The AAV9 capsid displays a high natural affinity for the heart following a single intravenous (IV) administration in both newborn and adult mice. It also results in substantial albeit relatively lower expression levels in many other tissues. To increase the overall safety of this gene delivery method we sought to identify which one of a group of promoters is able to confer the highest level of cardiac specific expression and concurrently, which is able to provide a broad biodistribution of expression across both cardiac and skeletal muscle. The in vivo behavior of five different promoters was compared: CMV, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC). Following IV administration to newborn mice, LacZ expression was measured by enzyme activity assays. Results showed that rAAV2/9-mediated gene delivery using the α-MHC promoter is effective for focal transgene expression in the heart and the Des promoter is highly suitable for achieving gene expression in cardiac and skeletal muscle following systemic vector administration. Importantly, these promoters provide an added layer of control over transgene activity following systemic gene delivery.     

Compatible solutes mitigate damaging effects of salt stress by reducing the impact of stress-induced reactive oxygen species

Under abiotic stress conditions, rapid increases in reactive oxygen species (ROS) levels occurs within plant cells. Although their role as a major signalling agent in plants is now acknowledged, elevated ROS levels can result in an impairment of membrane integrity. Similar to our previous findings on imposition of salt stress, application of the hydroxyl radical (OH^•) to Arabidopsis roots results in a massive efflux of K⁺ from epidermal cells. This is likely to cause significant damage to cell metabolism. Since K⁺ loss also occurs after salt application and salt stress leads to increased cellular ROS levels, we suggest that at least some of the detrimental effects of salinity is due to damage by its resulting ROS on K⁺ homeostasis. We also observed a comparative reduction in K⁺ efflux by compatible solutes after both oxidative and salt stress. Thus, we propose that under saline conditions, compatible solutes mitigate the oxidative stress damage to membrane transporters. Whether this amelioration is due to free-radical scavenging or by direct protection of transporter systems, warrants further investigation.Key words: compatible solutes, hydroxyl radical, potassium efflux, reactive oxygen species, salt stressReactive oxygen species (ROS) are continuously produced as by-products of various metabolic pathways.¹ Under unstressed steady-state conditions, cellular ROS levels are kept in check by the sophisticated antioxidant defence system.² However, under adverse environmental conditions, the balance between ROS production and its subsequent scavenging may be perturbed, leading to a rapid increase in ROS levels.³ Although significant progress has been made in defining ROS as a major signalling agent in plants,³ ROS can react with a large variety of biomolecules, causing lipid peroxidation and impairing membrane integrity.^4,5 One such abiotic stress is salt stress,⁶ with ROS generation occurring within minutes of salt application.⁷ Alleviation of oxidative damage may be, therefore, an important strategy of plant salt tolerance.⁸One of the earliest measurable responses to salt stress is a massive K⁺ efflux from plant roots.^9,10 Such K⁺ efflux is initiated within seconds of acute salt stress and may last for several hours^11,12 reducing the intracellular K⁺ pool^13,14 and significantly impairing cell metabolism. Consistent with the key role of K⁺ homeostasis in salt tolerance mechanisms¹⁵ a reduction of K⁺ efflux correlates with increased salt tolerance.^11,12We have previously reported that hydroxyl radical (OH^•) application to Arabidopsis roots also results in a rapid efflux of K⁺ from the epidermis.¹⁶ In this report, we find a similar K⁺ efflux response.¹⁷ As is the case for salt stress,⁹ we found that membrane depolarisation could be responsible for a substantial part of this efflux. However, an observed discrepancy between the membrane depolarisation and the pattern of K⁺ efflux indicates that voltage-dependence is not the only factor influencing K⁺ loss from the root cells after oxidative stress. Demidchik et al.¹⁶ demonstrated that stress-induced K⁺ efflux could be mediated by activation of K⁺ outward rectifying channels directly by OH^•. This direct effect on K⁺ transporters could also account for our observed delay before the peak efflux of K⁺ is measured, indicating that a certain amount of time is required before maximal direct damage by OH^• to transporters occurs. Because both K⁺ channel blockers and non-selective cation channel blockers reduce this efflux, it indicates non-specificity in OH^• attack. Furthermore, combinations of these channel blockers were effective in reducing K⁺ efflux implying that, at least in the short term, the damaging effects of OH^• is due to compromising the transporter systems as opposed to lipid peroxidation. Certainly, K⁺ channels harbour reactive groups, thus are expected to be sensitive to ROS.¹⁸We have previously shown that the exogenous application of low concentrations of a variety of compatible solutes reduces the salt-induced K⁺ efflux.^19,20 Plants, when confronted with a saline environment, respond with a significant elevation in their compatible solute levels. This ameliorates the detrimental effects of salinity.²¹ However, their original proposed role in cellular osmoregulation is under question: their concentration in transgenic plants overexpressing osmolyte biosynthetic genes is not significant for osmotic adjustment, despite showing improved salt tolerance.⁸ Furthermore, one hallmark of the detoxification effect is its lack of specificity, that is, transgenic plants have increased tolerance not only to high salt, but also to drought, cold and heat shock,^22,23 stresses that also result in ROS production.³ Certainly, ecotopic expression studies suggest that compatible solutes increase stress tolerance by protection of membranes and proteins against ROS.⁶We show that in this work that exogenous application of low concentrations of a range of compatible solutes significantly reduces OH^•-induced K⁺ efflux,¹⁷ a similar effect to that we reported after salt application to barley roots¹⁹ and also observed in Arabidopsis (Fig. 1). Interestingly, we found that not only known free-radical scavenging osmolytes,²⁴ but also glycine betaine, previously found to be non-effective in ROS scavenging,²⁴ were effective in reducing OH^•-induced K⁺ efflux. Indeed, glycine betaine showed a greater mitigation of OH^•-induced K⁺ efflux compared to that induced by 50 mM NaCl (Fig. 1). However, it is open to speculation as to whether this mitigation is via direct channel blocking, a direct protection of ion channel proteins or by some other protective mechanism.Open in a separate windowFigure 1Effects of exogenous supply of compatible solutes on net peak K⁺ efflux after application of either 1 mM Cu/a or 50 mM NaCl. Roots were preincubated for 1 h in 5 mM concentration of a number of compatible solutes prior to treatment, Mean ± SE (n = 6-8).In our further investigations we have found that salt-tolerant barley show a reduced ROS-induced K⁺ efflux compared to sensitive varieties.²⁵ This superior ability of salt-tolerant barley cultivars of preventing K⁺ loss further indicates a possible causal link between salt and oxidative stress tolerance. We propose that upon the imposition of salt stress, the instantaneously resulting membrane depolarisation9 results in activation of depolarisation activated K⁺ outward-rectifying channels, leading to the initial massive K⁺ efflux. Over the longer term, ROS levels within the plant cell increase,⁷ resulting in direct damage to K⁺ transporters and the longer-term sustained loss of K⁺ from the cell. Due to mitigation of both NaCl- and OH^•-induced K⁺ efflux by compatible solutes, we propose that one of their primary amelioratory effects is through reducing the damaging effects of salt-produced ROS on K⁺ transporter, and by this means, reducing the effects of stress damage. Whether this amelioration is achieved through free-radical scavenging or due to a direct protection of membrane transports warrants further investigation.     

Cofilin activation in peripheral CD4 T cells of HIV-1 infected patients: a pilot study

The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.     

Development of microsatellite markers in Cordia bifurcata (Boraginaceae) and cross-species amplification in Cordia inermis and Cordia pringlei

We developed 16 microsatellite markers in Cordia bifurcata, a Central and South American shrub. The markers show low polymorphism in C. bifurcata, a species suspected of self-fertilization or apomixis. Of four polymorphic loci, three had only two alleles. However, current research indicates that these markers hold value for interpopulational comparisons of C. bifurcata and for analyses of congeners. In Cordia inermis, a dioecious or subdioecious shrub, seven of the markers produced interpretable amplification products of which five showed polymorphism. In Cordia pringlei, a distylous shrub, nine of the markers produced interpretable amplification products of which six showed polymorphism.