Mutations in the TΨC Loop of E. coli tRNA(Lys,3 )Have Varied Effects on In Trans Complementation of HIV-1 Replication

BACKGROUND: Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNA(Lys,3 )as the primer for initiation of reverse transcription. Several elements within the TΨC stem loop of tRNA(Lys,3 )are postulated to be important for selection and use in reverse transcription. The post-transcriptional modification at nucleotide 58 could play a role during plus-strand synthesis to stop reverse transcriptase from re-copying the tRNA primer. Nucleotides 53 and 54 within the TΨC stem loop of the tRNA have been shown to be important to form the complex between tRNA and the HIV-1 viral genome during initiation of reverse transcription. RESULTS: To further delineate the features of the TΨC stem loop of tRNA(Lys,3 )in reverse transcription, we have developed a complementation system in which E. coli tRNA(Lys,3 )is provided in trans to an HIV-1 genome in which the PBS is complementary to this tRNA. Successful selection and use of E. coli tRNA(Lys,3 )results in the production of infectious virus. We have used this single round infectious system to ascertain the effects that different mutants in the TΨC stem loop of tRNA(Lys,3 )have on complementation. Mutants were designed within the TΨC loop (nucleotide 58) and within the stem and loop of the TΨC loop (nucleotides 53 and 54). Analysis of the expression of E. coli tRNA(Lys,3 )mutants revealed differences in the capacity for aminoacylation, which is an indication of intracellular stability of the tRNA. Alteration of nucleotide 58 from A to U (A58U), T54G and TG5453CC all resulted in tRNA(Lys,3 )that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNA(Lys,3 )that was not aminoacylated. Both the A58U and T54G mutated tRNA(Lys,3 )complemented HIV-1 replication similar to wild type E. coli tRNA(Lys,3). In contrast, the TG5453CC tRNA(Lys,3 )mutant did not complement replication. CONCLUSION: The results demonstrate that post-transcriptional modification of nucleotide 58 in tRNA(Lys,3 )is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNA(Lys,3 )are important for aminoacylation and selection and use of the tRNA(Lys,3 )in reverse transcription

Warming Reduces Tall Fescue Abundance but Stimulates Toxic Alkaloid Concentrations in Transition Zone Pastures of the U.S.

Tall fescue pastures cover extensive acreage in the eastern half of the United States and contribute to important ecosystem services, including the provisioning of forage for grazing livestock. Yet little is known concerning how these pastures will respond to climate change. Tall fescue\u27s ability to persist and provide forage under a warmer and wetter environment, as is predicted for much of this region as a result of climate change, will likely depend on a symbiotic relationship the plant can form with the fungal endophyte, Epichloë coenophiala. While this symbiosis can confer environmental stress tolerance to the plant, the endophyte also produces alkaloids toxic to insects (e.g., lolines) and mammals (ergots; which can cause fescue toxicosis in grazing animals). The negative animal health and economic consequences of fescue toxicosis make understanding the response of the tall fescue symbiosis to climate change critical for the region. We experimentally increased temperature (+3°C) and growing season precipitation (+30% of the long-term mean) from 2009-2013 in a mixed species pasture, that included a tall fescue population that was 40% endophyte-infected. Warming reduced the relative abundance of tall fescue within the plant community, and additional precipitation did not ameliorate this effect. Warming did not alter the incidence of endophyte infection within the tall fescue population; however, warming significantly increased concentrations of ergot alkaloids (by 30-40%) in fall-harvested endophyte-infected individuals. Warming alone did not affect loline alkaloid concentrations, but when combined with additional precipitation, levels increased in fall-harvested material. Although future warming may reduce the dominance of tall fescue in eastern U.S. pastures and have limited effect on the incidence of endophyte infection, persisting endophyte-infected tall fescue will have higher concentrations of toxic alkaloids which may exacerbate fescue toxicosis

Vpx is Critical for SIVmne infection of pigtail macaques

<p>Abstract</p> <p>Background</p> <p>Viral protein X (Vpx) of SIV has been reported to be important for establishing infection <it>in vivo</it>. Vpx has several different activities <it>in vitro</it>, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important <it>in vivo</it>.</p> <p>Results</p> <p>An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions <it>in vitro </it>and <it>in vivo</it>. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A <it>vpx</it>-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the <it>vpx </it>mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the <it>vpx </it>mutant viruses. <it>In vitro</it>, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three <it>vpx </it>mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the <it>vpx </it>mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. <it>In situ </it>hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the <it>vpx </it>mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.</p> <p>Conclusions</p> <p>This work demonstrates that the activities of Vpx to overcome restrictions in culture <it>in vitro </it>are also likely to be important for establishment of infection <it>in vivo </it>and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical <it>in vivo</it>.</p

Nitrogen but not phosphorus addition affects symbiotic N2 fixation by legumes in natural and semi‑natural grasslands located on four continents

The amount of nitrogen (N) derived from symbiotic N2 fixation by legumes in grasslands might be affected by anthropogenic N and phosphorus (P) inputs, but the underlying mechanisms are not known. Methods We evaluated symbiotic N2 fixation in 17 natural and semi-natural grasslands on four continents that are subjected to the same full-factorial N and P addition experiment, using the 15N natural abundance method. Results N as well as combined N and P (NP) addition reduced aboveground legume biomass by 65% and 45%, respectively, compared to the control, whereas P addition had no significant impact. Addition of N and/or P had no significant effect on the symbiotic N2 fixation per unit legume biomass. In consequence, the amount of N fixed annually per grassland area was less than half in the N addition treatments compared to control and P addition, irrespective of whether the dominant legumes were annuals or perennials. Conclusion Our results reveal that N addition mainly impacts symbiotic N2 fixation via reduced biomass of legumes rather than changes in N2 fixation per unit legume biomass. The results show that soil N enrichment by anthropogenic activities significantly reduces N 2 fixation in grasslands, and these effects cannot be reversed by additional P amendment.EEA Santa CruzFil: Vázquez, Eduardo. University of Bayreuth. Department of Soil Ecology. Bayreuth Center of Ecology and Environmental Research (BayCEER); AlemaniaFil: Vázquez, Eduardo. Swedish University of Agricultural Sciences. Department of Soil and Environment; SueciaFil: Schleuss, Per‑Marten. University of Bayreuth. Department of Soil Ecology. Bayreuth Center of Ecology and Environmental Research (BayCEER); AlemaniaFil: Borer, Elizabeth T. University of Minnesota. Department of Ecology, Evolution, and Behavior; Estados UnidosFil: Bugalho, Miguel N. University of Lisbon. Centre for Applied Ecology “Prof. Baeta Neves” (CEABN-InBIO). School of Agriculture; Portugal.Fil: Caldeira, Maria. C. University of Lisbon. Forest Research Centre. School of Agriculture; Portugal.Fil: Eisenhauer, Nico. German Centre for Integrative Biodiversity Research; AlemaniaFil: Eisenhauer, Nico. Leipzig University. Institute of Biology; AlemaniaFil: Eskelinen, Anu. German Centre for Integrative Biodiversity Research; AlemaniaFil: Eskelinen, Anu. Physiological Diversity, Helmholtz Centrefor Environmental Research; AlemaniaFil: Eskelinen, Anu. University of Oulu. Ecology & Genetics; FinlandiaFil: Fay, Philip A. Grassland Soil and Water Research Laboratory (USDA-ARS); Estados UnidosFil: Haider, Sylvia. German Centre for Integrative Biodiversity Research; AlemaniaFil: Haider, Sylvia. Martin Luther University. Institute of Biology. Geobotany and Botanical Garden; AlemaniaFil: Jentsch, Anke. University of Bayreuth. Department of Soil Ecology. Bayreuth Center of Ecology and Environmental Research (BayCEER); AlemaniaFil: Kirkman, Kevin P. University of KwaZulu-Natal. School of Life Sciences; SudáfricaFil: McCulley, Rebecca L. University of Kentucky. Department of Plant and Soil Sciences; Estados UnidosFil: Peri, Pablo Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Santa Cruz; Argentina.Fil: Peri, Pablo Luis. Universidad Nacional de la Patagonia Austral; Argentina.Fil: Peri, Pablo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Price, Jodi. Charles Sturt University. Institute for Land, Water and Society; Australia.Fil: Richards, Anna E. CSIRO Land and Water. Northern Territory; Australia.Fil: Risch, Anita C. Swiss Federal Institute for Forest, Snow and Landscape Research WSL; SuizaFil: Roscher, Christiane. German Centre for Integrative Biodiversity Research; AlemaniaFil: Roscher, Christiane. Physiological Diversity, Helmholtz Centre for Environmental Research; AlemaniaFil: Schütz, Martin. Swiss Federal Institute for Forest, Snow and Landscape Research WSL; SuizaFil: Seabloom, Eric William. University of Minnesota. Dept. of Ecology, Evolution, and Behavior; Estados UnidosFil: Standish, Rachel J. Murdoch University. Harry Butler Institute; Australia.Fil: Stevens, Carly J. Lancaster University. Lancaster Environment Centre; Reino UnidoFil: Tedder, Michelle J. University of KwaZulu-Natal. School of Life Sciences; SudáfricaFil: Virtanen, Risto. University of Oulu. Ecology & Genetics; Finlandia.Fil: Spohn, Marie. University of Bayreuth. Department of Soil Ecology. Bayreuth Center of Ecology and Environmental Research (BayCEER); AlemaniaFil: Spohn, Marie. Swedish University of Agricultural Sciences. Department of Soil and Environment; Sueci

Nitrogen but not phosphorus addition affects symbiotic N-2 fixation by legumes in natural and semi-natural grasslands located on four continents

Background and aims: The amount of nitrogen (N) derived from symbiotic N-2 fixation by legumes in grasslands might be affected by anthropogenic N and phosphorus (P) inputs, but the underlying mechanisms are not known.Methods: We evaluated symbiotic N-2 fixation in 17 natural and semi-natural grasslands on four continents that are subjected to the same full-factorial N and P addition experiment, using the N-15 natural abundance method.Results: N as well as combined N and P (NP) addition reduced aboveground legume biomass by 65% and 45%, respectively, compared to the control, whereas P addition had no significant impact. Addition of N and/or P had no significant effect on the symbiotic N-2 fixation per unit legume biomass. In consequence, the amount of N fixed annually per grassland area was less than half in the N addition treatments compared to control and P addition, irrespective of whether the dominant legumes were annuals or perennials.Conclusion: Our results reveal that N addition mainly impacts symbiotic N-2 fixation via reduced biomass of legumes rather than changes in N-2 fixation per unit legume biomass. The results show that soil N enrichment by anthropogenic activities significantly reduces N-2 fixation in grasslands, and these effects cannot be reversed by additional P amendment

Plant Species\u27 Origin Predicts Dominance and Response to Nutrient Enrichment and Herbivores in Global Grasslands

Exotic species dominate many communities; however the functional significance of species\u27 biogeographic origin remains highly contentious. This debate is fuelled in part by the lack of globally replicated, systematic data assessing the relationship between species provenance, function and response to perturbations. We examined the abundance of native and exotic plant species at 64 grasslands in 13 countries, and at a subset of the sites we experimentally tested native and exotic species responses to two fundamental drivers of invasion, mineral nutrient supplies and vertebrate herbivory. Exotic species are six times more likely to dominate communities than native species. Furthermore, while experimental nutrient addition increases the cover and richness of exotic species, nutrients decrease native diversity and cover. Native and exotic species also differ in their response to vertebrate consumer exclusion. These results suggest that species origin has functional significance, and that eutrophication will lead to increased exotic dominance in grasslands

Negative effects of nitrogen override positive effects of phosphorus on grassland legumes worldwide

Anthropogenic nutrient enrichment is driving global biodiversity decline and modifying ecosystem functions. Theory suggests that plant functional types that fix atmospheric nitrogen have a competitive advantage in nitrogen-poor soils, but lose this advantage with increasing nitrogen supply. By contrast, the addition of phosphorus, potassium, and other nutrients may benefit such species in low-nutrient environments by enhancing their nitrogen-fixing capacity. We present a global-scale experiment confirming these predictions for nitrogen-fixing legumes (Fabaceae) across 45 grasslands on six continents. Nitrogen addition reduced legume cover, richness, and biomass, particularly in nitrogen-poor soils, while cover of non–nitrogen-fixing plants increased. The addition of phosphorous, potassium, and other nutrients enhanced legume abundance, but did not mitigate the negative effects of nitrogen addition. Increasing nitrogen supply thus has the potential to decrease the diversity and abundance of grassland legumes worldwide regardless of the availability of other nutrients, with consequences for biodiversity, food webs, ecosystem resilience, and genetic improvement of protein-rich agricultural plant species.DATA AVAILABILITY : Plant, PAR, climate, and soil nitrogen data have been deposited in the Environmental Data Initiative (EDI) repository (https://portal.edirepository.org/nis/mapbrowse?packageid=edi.838.1) (83). Source data are provided with this paper.This work was generated using data from the Nutrient Network (https://nutnet.org/) experiment, funded at the site scale by individual researchers. Coordination and data management were supported by funding to E.T.B. and E.W.S. from the NSF Research Coordination Network (NSF-DEB-1042132) and Long-Term Ecological Research (NSF-DEB-1234162 to Cedar Creek Long-Term Ecological Research) programs, and the Institute on the Environment (DG-0001-13). We also thank the Minnesota Supercomputer Institute for hosting project data and the Institute of the Environment for hosting Network meetings. P.M.T. was supported by an Argentine Research Council fellowship (Consejo Nacional de Investigaciones Científicas y Técnicas) and the Australian Endeavour Programme.https://www.pnas.orghj2022Mammal Research InstituteZoology and Entomolog

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

Widening global variability in grassland biomass since the 1980s

Global change is associated with variable shifts in the annual production of aboveground plant biomass, suggesting localized sensitivities with unclear causal origins. Combining remotely sensed normalized difference vegetation index data since the 1980s with contemporary field data from 84 grasslands on 6 continents, we show a widening divergence in site-level biomass ranging from +51% to −34% globally. Biomass generally increased in warmer, wetter and species-rich sites with longer growing seasons and declined in species-poor arid areas. Phenological changes were widespread, revealing substantive transitions in grassland seasonal cycling. Grazing, nitrogen deposition and plant invasion were prevalent in some regions but did not predict overall trends. Grasslands are undergoing sizable changes in production, with implications for food security, biodiversity and carbon storage especially in arid regions where declines are accelerating

Plant species' origin predicts dominance and response to nutrient enrichment and herbivores in global grasslands

Exotic species dominate many communities; however the functional significance of species’ biogeographic origin remains highly contentious. This debate is fuelled in part by the lack of globally replicated, systematic data assessing the relationship between species provenance, function and response to perturbations. We examined the abundance of native and exotic plant species at 64 grasslands in 13 countries, and at a subset of the sites we experimentally tested native and exotic species responses to two fundamental drivers of invasion, mineral nutrient supplies and vertebrate herbivory. Exotic species are six times more likely to dominate communities than native species. Furthermore, while experimental nutrient addition increases the cover and richness of exotic species, nutrients decrease native diversity and cover. Native and exotic species also differ in their response to vertebrate consumer exclusion. These results suggest that species origin has functional significance, and that eutrophication will lead to increased exotic dominance in grasslands