The HIV-1 Integrase Mutations Y143C/R Are an Alternative Pathway for Resistance to Raltegravir and Impact the Enzyme Functions

Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3′end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3′end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3′end-processing assay, IC₅₀ were 0.4 µM, 0.9 µM (FC = 2.25) and 1.2 µM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3′P but mutants were more resistant to RAL than WT.     

Molecular characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and impact of T-cell epitope mutations on HLA recognition (ANRS 12159)

Background

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.

Methods

We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score).

Results

All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).

Conclusions

Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area.     

Mixing Eucalyptus and Acacia trees leads to fine root over-yielding and vertical segregation between species

The consequences of diversity on belowground processes are still poorly known in tropical forests. The distributions of very fine roots (diameter <1 mm) and fine roots (diameter <3 mm) were studied in a randomized block design close to the harvest age of fast-growing plantations. A replacement series was set up in Brazil with mono-specific Eucalyptus grandis (100E) and Acacia mangium (100A) stands and a mixture with the same stocking density and 50 % of each species (50A:50E). The total fine root (FR) biomass down to a depth of 2 m was about 27 % higher in 50A:50E than in 100A and 100E. Fine root over-yielding in 50A:50E resulted from a 72 % rise in E. grandis fine root biomass per tree relative to 100E, whereas A. mangium FR biomass per tree was 17 % lower than in 100A. Mixing A. mangium with E. grandis trees led to a drop in A. mangium FR biomass in the upper 50 cm of soil relative to 100A, partially balanced by a rise in deep soil layers. Our results highlight similarities in the effects of directional resources on leaf and FR distributions in the mixture, with A. mangium leaves below the E. grandis canopy and a low density of A. mangium fine roots in the resource-rich soil layers relative to monospecific stands. The vertical segregation of resource-absorbing organs did not lead to niche complementarity expected to increase the total biomass production.     

Measured and modeled interactive effects of potassium deficiency and water deficit on gross primary productivity and light‐use efficiency in Eucalyptus grandis plantations

Global climate change is expected to increase the length of drought periods in many tropical regions. Although large amounts of potassium (K) are applied in tropical crops and planted forests, little is known about the interaction between K nutrition and water deficit on the physiological mechanisms governing plant growth. A process‐based model (MAESPA) parameterized in a split‐plot experiment in Brazil was used to gain insight into the combined effects of K deficiency and water deficit on absorbed radiation (aPAR), gross primary productivity (GPP), and light‐use efficiency for carbon assimilation and stem biomass production (LUE_C and LUE_s) in Eucalyptus grandis plantations. The main‐plot factor was the water supply (undisturbed rainfall vs. 37% of throughfall excluded) and the subplot factor was the K supply (with or without 0.45 mol K m^?2 K addition). Mean GPP was 28% lower without K addition over the first 3 years after planting whether throughfall was partly excluded or not. K deficiency reduced aPAR by 20% and LUE_C by 10% over the whole period of growth. With K addition, throughfall exclusion decreased GPP by 25%, resulting from a 21% decrease in LUE_C at the end of the study period. The effect of the combination of K deficiency and water deficit was less severe than the sum of the effects of K deficiency and water deficit individually, leading to a reduction in stem biomass production, gross primary productivity and LUE similar to K deficiency on its own. The modeling approach showed that K nutrition and water deficit influenced absorbed radiation essentially through changes in leaf area index and tree height. The changes in gross primary productivity and light‐use efficiency were, however, driven by a more complex set of tree parameters, especially those controlling water uptake by roots and leaf photosynthetic capacities.     

Rapamycin‐mediated mTORC2 inhibition is determined by the relative expression of FK506‐binding proteins

The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age‐related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506‐binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin‐mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general.     

Reconstruction and attribution of the carbon sink of European forests between 1950 and 2000

European forests are an important carbon sink; however, the relative contributions to this sink of climate, atmospheric CO₂ concentration ([CO₂]), nitrogen deposition and forest management are under debate. We attributed the European carbon sink in forests using ORCHIDEE‐FM, a process‐based vegetation model that differs from earlier versions of ORCHIDEE by its explicit representation of stand growth and idealized forest management. The model was applied on a grid across Europe to simulate changes in the net ecosystem productivity (NEP) of forests with and without changes in climate, [CO₂] and age structure, the three drivers represented in ORCHIDEE‐FM. The model simulates carbon stocks and volume increment that are comparable – root mean square error of 2 m³ ha^?1 yr^?1 and 1.7 kg C m^?2 respectively – with inventory‐derived estimates at country level for 20 European countries. Our simulations estimate a mean European forest NEP of 175 ± 52 g C m^?2 yr^?1 in the 1990s. The model simulation that is most consistent with inventory records provides an upwards trend of forest NEP of 1 ± 0.5 g C m^?2 yr^?2 between 1950 and 2000 across the EU 25. Furthermore, the method used for reconstructing past age structure was found to dominate its contribution to temporal trends in NEP. The potentially large fertilizing effect of nitrogen deposition cannot be told apart, as the model does not explicitly simulate the nitrogen cycle. Among the three drivers that were considered in this study, the fertilizing effect of increasing [CO₂] explains about 61% of the simulated trend, against 26% to changes in climate and 13% only to changes in forest age structure. The major role of [CO₂] at the continental scale is due to its homogeneous impact on net primary productivity (NPP). At the local scale, however, changes in climate and forest age structure often dominate trends in NEP by affecting NPP and heterotrophic respiration.     

Leaf respiration is differentially affected by leaf vs. stand-level night-time warming

Plant respiration is an important physiological process in the global carbon cycle serving as a major carbon flux from the biosphere to the atmosphere. Respiration is sensitive to temperature providing a link between environmental variability, climate change and the global carbon cycle. We measured leaf respiration in Populus deltoides after manipulating the air temperature surrounding part of a single leaf, and compared this to the temperature response of the same leaves after manipulating the temperature of the stand. The short‐term temperature response of respiration (Q₁₀– change in the respiration rate with a 10 °C increase in leaf temperature) was 1.7 when the leaf temperature was manipulated, but 2.1 when the stand‐level temperature was changed. As a result, total night‐time carbon release during the five‐day experiment was 21% lower when using the Q₁₀ estimates from the tradition leaf manipulation compared to the stand‐level manipulation. We conclude that the temperature response of leaf respiration is related to whole plant carbon and energy demands, and that appropriate experimental procedures are required in examining respiratory CO₂ release under variable temperature conditions.     

Age-related changes in litter inputs explain annual trends in soil CO2 effluxes over a full Eucalyptus rotation after afforestation of a tropical savannah

Land use changes such as savannah afforestation with eucalypts impact the soil carbon (C) balance, therefore affecting soil CO₂ efflux (F _s ), a major flux in the global C cycle. We tested the hypothesis that F _s increases with stand age after afforestation, due to an increasing input of fresh organic matter to the forest floor. In a Eucalyptus plantation established on coastal savannahs in Congo, bimonthly measurements of F _s were carried out for 1 year on three adjacent stands aged 0.9, 4.4 and 13.7 years and presenting similar growth patterns. Litterfall and litter accumulation on the forest floor were quantified over a chronosequence. Equations were derived to estimate the contribution of litter decomposition to F _s throughout the rotation. Litterfall increased with stand age after savannah afforestation. F _s , that was strongly correlated on a seasonal basis with soil water content (SWC) in all stands, decreased between ages 0.9 year and 4.4 years due to savannah residue depletion, and increased between ages 4.4 years and 13.7 years, mainly because of an increasing amount of decomposing eucalypt litter. The aboveground litter layer therefore appeared as a major source of CO₂, whose contribution to F _s in old stands was estimated to be about four times higher than that of the eucalypt-derived soil organic C pool. The high litter contribution to F _s in older stands might explain why 13.7 years-old stand F _s was limited by moisture all year round whereas SWC did not limit F _s for large parts of the year in the youngest stands.     

Increased light‐use efficiency sustains net primary productivity of shaded coffee plants in agroforestry system

In agroforestry systems, shade trees strongly affect the physiology of the undergrown crop. However, a major paradigm is that the reduction in absorbed photosynthetically active radiation is, to a certain extent, compensated by an increase in light‐use efficiency, thereby reducing the difference in net primary productivity between shaded and non‐shaded plants. Due to the large spatial heterogeneity in agroforestry systems and the lack of appropriate tools, the combined effects of such variables have seldom been analysed, even though they may help understand physiological processes underlying yield dynamics. In this study, we monitored net primary productivity, during two years, on scales ranging from individual coffee plants to the entire plot. Absorbed radiation was mapped with a 3D model (MAESPA). Light‐use efficiency and net assimilation rate were derived for each coffee plant individually. We found that although irradiance was reduced by 60% below crowns of shade trees, coffee light‐use efficiency increased by 50%, leaving net primary productivity fairly stable across all shade levels. Variability of aboveground net primary productivity of coffee plants was caused primarily by the age of the plants and by intraspecific competition among them (drivers usually overlooked in the agroforestry literature) rather than by the presence of shade trees.     

Cdc42-driven podosome formation in endothelial cells

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.