Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who control viremia to levels below the limit of detection of current assays. The mechanisms involved in this control have not been fully elucidated. Several studies have demonstrated that some ES are infected with defective viruses, but it remains unclear whether others are infected with replication-competent HIV-1. To answer this question, we used a sensitive coculture assay in an attempt to isolate replication-competent virus from a cohort of 10 ES. We successfully cultured six replication-competent isolates from 4 of the 10 ES. The frequency of latently infected cells in these patients was more than a log lower than that seen in patients on highly active antiretroviral therapy with undetectable viral loads. Full-length sequencing of all six isolates revealed no large deletions in any of the genes. A few mutations and small insertions and deletions were found in some isolates, but phenotypic analysis of the affected genes suggested that their function remained intact. Furthermore, all six isolates replicated as well as standard laboratory strains in vitro. The results suggest that some ES are infected with HIV-1 isolates that are fully replication competent and that long-term immunologic control of replication-competent HIV-1 is possible.     

Chloroplast and nuclear microsatellite analysis of Aegilops cylindrica

Aegilops cylindrica Host (2n=4x=28, genome CCDD) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n=2x=14, genome DD) and Ae. markgrafii (Greuter) Hammer (2n=2x=14, genome CC). Previous research has shown that Ae. tauschii contributed its cytoplasm to Ae. cylindrica. However, our analysis with chloroplast microsatellite markers showed that 1 of the 36 Ae. cylindrica accessions studied, TK 116 (PI 486249), had a plastome derived from Ae. markgrafii rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its cytoplasm to Ae. cylindrica. Our analysis of chloroplast and nuclear microsatellite markers also suggests that D-type plastome and the D genome in Ae. cylindrica were closely related to, and were probably derived from, the tauschii gene pool of Ae. tauschii. A determination of the likely source of the C genome and the C-type plastome in Ae. cylindrica was not possible.     

Community-Level Physiological Profiles of Cloacal Microbes in Songbirds (Order: Passeriformes): Variation Due to Host Species, Host Diet, and Habitat

The relationship between microorganisms and birds has received increased attention recently. The state of knowledge of this relationship, however, is based largely on examination of sick or dead birds, and knowledge of the prevalence and community structure and function of microbes in healthy wild populations is limited. Using carbon substrate utilization profiles, microbial communities were examined in 91 cloacal samples from 14 species within apparently healthy summer and winter passerine populations. Within each season, gradient lengths and eigenvalues from ordination analyses suggested that many samples differed in their carbon substrate utilization and several had very different communities. Cloacal microbe carbon utilization profiles were distinguishable among host species, season-specific diet, and study site in the ordination analyses. However, these patterns were only observed for the analysis of the summer data set. The results of this study support the idea that the avian host’s microbial community, relative to carbon substrate utilization, is related to host diet. Previously, this pattern had only been reported for potential pathogens isolated from the avian cloaca. Study site–specific patterns in the ordination analysis suggest that environmental conditions at a particular study site may influence cloacal microbial communities in birds. Results of this study indicate that examination of community-level physiological profiles may be a useful technique for distinguishing among avian cloacal samples, similar to that already established for discriminating aqueous and soil samples. Future studies that correlate microbe physiological profiles to condition-based indices of avian hosts may be most useful for eventually using the profile as an indicator of environmental conditions experienced by hosts.     

Population genetic approaches to neurological disease: Parkinson's disease as an example

Parkinson's disease (PD) is a common, progressive, incurable disabling condition. The cause is unknown but over the past few years tremendous progress in our understanding of the genetic bases of this condition has been made. To date, this has almost exclusively come from the study of relatively rare Mendelian forms of the disease and there are no currently, widely accepted common variants known to increase susceptibility.The role that the "Mendelian" genes play in common sporadic forms of PD is unknown. Moreover, most studies in PD can really be described as candidate polymorphism studies rather than true and complete assessments of the genes themselves. We provide a model of how one might tackle some of these issues using Parkinson's disease as an illustration. One of the emerging hypotheses of gene environment interaction in Parkinson's disease is based on drug metabolizing (or xenobiotic) enzymes and their interaction with putative environmental toxins. This motivated us to describe a tagging approach for an extensive but not exhaustive list of 55 drug metabolizing enzyme genes. We use these data to illustrate the power, and some of the limitations of a haplotype tagging approach. We show that haplotype tagging is extremely efficient and works well with only a modest increase in effort through different populations. The tagging approach works much less well if the minor allele frequency is below 5%. However, it will now be possible using these tags to evaluate these genes comprehensively in PD and other neurodegenerative conditions.     

Dopamine Induces Ca2+ Signaling in Astrocytes through Reactive Oxygen Species Generated by Monoamine Oxidase

Dopamine is a neurotransmitter that plays a major role in a variety of brain functions, as well as in disorders such as Parkinson disease and schizophrenia. In cultured astrocytes, we have found that dopamine induces sporadic cytoplasmic calcium ([Ca²⁺]_c) signals. Importantly, we show that the dopamine-induced calcium signaling is receptor-independent in midbrain, cortical, and hippocampal astrocytes. We demonstrate that the calcium signal is initiated by the metabolism of dopamine by monoamine oxidase, which produces reactive oxygen species and induces lipid peroxidation. This stimulates the activation of phospholipase C and subsequent release of calcium from the endoplasmic reticulum via the inositol 1,4,5-trisphosphate receptor mechanism. These findings have major implications on the function of astrocytes that are exposed to dopamine and may contribute to understanding the physiological role of dopamine.     

Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

Apoptotic cells are considered an important auto-antigenic source in diseases such as systemic lupus erythematosus (SLE). A human monoclonal antibody demonstrating exquisite specificity towards late-stage apoptotic cells was generated from an SLE patient. Polyreactive recognition of ribonucleoproteins Ro52 and Ro60 was observed. The antibody significantly diminished the phagocytosis of apoptotic cells and a concomitant decrease in transforming growth factor-beta (TGF-beta) secretion was observed. Light and heavy chain sequencing revealed the antibody to be in essentially germline configuration. Elicited anti-idiotypic antibodies bound distinct self-antigens and showed augmented reactivity towards apoptotic cells as well. Thus, near-germline encoded antibodies recognizing antigens externalized during the process of apoptosis can mediate a variety of potentially pathogenic effects; decreases in the phagocytic uptake of dying cells would constitute a disease-perpetuating event and stimulation of the idiotypic network could lead to intermolecular epitope spreading, increasing the range of molecular targets..     

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome

Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.     

Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+ CD25- LAP+ T cells

A major goal of immunotherapy for autoimmune diseases and transplantation is induction of regulatory T cells that mediate immunologic tolerance. The mucosal immune system is unique, as tolerance is preferentially induced after exposure to antigen, and induction of regulatory T cells is a primary mechanism of oral tolerance. Parenteral administration of CD3-specific monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. We found that orally administered CD3-specific antibody is biologically active in the gut and suppresses autoimmune encephalomyelitis both before induction of disease and at the height of disease. Orally administered CD3-specific antibody induces CD4+ CD25- LAP+ regulatory T cells that contain latency-associated peptide (LAP) on their surface and that function in vitro and in vivo through a TGF-beta-dependent mechanism. These findings identify a new immunologic approach that is widely applicable for the treatment of human autoimmune conditions.     

Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA₂₀ and GA₈, in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.Semidwarfism is a valuable trait in many crop species and agricultural environments. In cereal crops, it can result in decreased lodging, increased yield, and improved harvest index (Dalrymple, 1985; Hedden, 2003). Therefore, it was a critical foundation of the “Green Revolution” that resulted in large improvements of yield in wheat (Triticum aestivum) and rice (Oryza sativa; Hargrove and Cabanilla, 1979; Perovic et al., 2008). Semidwarfism has had substantial benefits for fruit tree production, where it enables earlier fruit bearing, higher yields, and easier harvests in orchards (Battisini and Battisini, 2005). Semidwarf woody species are also extensively used in ornamental horticulture, where they allow more compact forms to be fit into small areas around homes and on streets and reduce the need for pruning to avoid interference with structures and transmission lines (Busov et al., 2003).Although against the current orthodoxy of forest tree breeding, where height growth is emphasized, semidwarfism might also have benefits for wood and biomass production (Bradshaw and Strauss, 2001). Such trees could be useful if they were less prone to wind throw due to their shorter, stockier forms and expected greater allocation to roots. Reduced stature could also result in less bending and slanting of trunks in the face of wind and gravity on hillslopes and thus reduce the extent of reaction wood formation, which degrades the performance and value of solid wood and pulp products. Reduced height and increased allocation of growth to roots might enhance stress tolerance, soil nutrient uptake, bioremediation, and carbon sequestration.Semidwarfism can be achieved by the modification of several types of genes and physiological mechanisms, but the most prevalent and advanced forms in agriculture affect GAs or their signaling (for review, see Busov et al., 2008). GAs are endogenous plant hormones that influence several aspects of plant growth and development, including seed germination, leaf expansion, shoot growth, cell division, flower induction, and fruit development (Sun and Gubler, 2004; Fleet and Sun, 2005; Swain and Singh, 2005). With respect to shoot growth, the most obvious effect of GA is its promotion of stem elongation by stimulating both cell elongation and division (Marth et al., 1956). GA modification also has significant effects on plant biochemistry, changing the amounts and distribution of a wide variety of metabolites in shoots and roots (Rossetto et al., 2003; Chen et al., 2004; Busov et al., 2006).Little is known about how semidwarfism affects belowground growth. GA has been shown to play a controlling role in lateral root development (Gou et al., 2010), and GA and ethylene synergistically promote both the initiation and growth of adventitious roots (Osmont et al., 2007). In tomato (Solanum lycopersicum), isogenic GA-deficient mutants (gib) allocate more biomass to roots compared with shoots (Nagel et al., 2001). In poplar (Populus spp.), semidwarf transgenic plants grown in vitro had a lower shoot-to-root ratio, which was at least partly due to proliferation of lateral roots (Busov et al., 2006; Gou et al., 2010).As a domestication trait, semidwarfism has been proposed as a means for mitigating the spread of transgenic plants within and outside of crop environments (Al-Ahmad et al., 2005). The genetic dominance of most semidwarfism transgenes would cause reduced height growth in transgene-containing progeny, reducing their ability to compete for light. Moreover, because of the close linkage of the semidwarfism genes to other genes that were cointroduced on the same plasmid, they would also powerfully retard their spread or introgression, even in cases where the linked transgene would, on their own, impart a selective advantage. However, there have been very few plant species where this concept has been explicitly tested (Al-Ahmad and Gressel, 2006; Gressel and Valverde, 2009), and we know of no examples in woody or perennial plants.To study the effects of semidwarfism genes in a woody plant grown under field conditions, we inserted a number of dominant GA-modifying transgenes into hybrid poplar (Populus tremula × Populus alba), the widely recognized model woody plant for genomics and biotechnology (Herschbach and Kopriva, 2002; Brunner et al., 2004a; Tuskan et al., 2004). Most of the genes studied were overexpressed forms of GA 2-oxidase, GA-Insensitive (GAI), or Repressor of GAI-Like (RGL), all known to cause semidwarfism in other plant species. GA 2-oxidase is a major GA catabolic enzyme in plants, and GAI and RGL are negative regulators of the GA signal transduction pathway (Appleford et al., 2007; Busov et al., 2008). The transgenic trees were first analyzed in the greenhouse (Busov et al., 2006) and then assayed for their effect on height growth in a 2-year field trial (Zawaski et al., 2011), from which we selected 10 transgenic events that grew at approximately three-quarters the rate of wild-type trees. The goal was to select semidwarf trees whose phenotype was not so severe as to be irrelevant to possible crop uses but strong enough to give a clear phenotype in a field study. In this study, we analyzed changes in a number of morphological, physiological, and growth traits and investigated the prospect for semidwarfism to be used as a mitigation tool to reduce the frequency of spread of transgenic and exotic species.