S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

Clinical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than children

Background

As dengue spreads to new geographical regions and the force of infection changes in existing endemic areas, a greater breadth of clinical presentations is being recognised. Clinical experience suggests that adults manifest a pattern of complications different from those observed in children, but few reports have described the age-related spectrum of disease in contemporaneous groups of patients recruited at the same geographical location.

Methodology/Principal Findings

Using detailed prospectively collected information from ongoing studies that encompass the full spectrum of hospitalised dengue cases admitted to a single hospital in southern Vietnam, we compared clinical and laboratory features, management, and outcome for 647 adults and 881 children with confirmed dengue. Signs of vascular leakage and shock were more frequent and more severe in children than adults, while bleeding manifestations and organ involvement were more common in adults. Additionally, adults experienced significantly more severe thrombocytopenia. Secondary infection but not serotype was independently associated with greater thrombocytopenia, although with a smaller effect than age-group. The effect of age-group on platelet count was also apparent in the values obtained several weeks after recovery, indicating that healthy adults have intrinsically lower counts compared to children.

Conclusions/Significance

There are clear distinctions between adults and children in the pattern of complications seen in association with dengue infection, and these depend partly on intrinsic age-dependent physiological differences. Knowledge of such differences is important to inform research on disease pathogenesis, as well as to encourage development of management guidelines that are appropriate to the age-groups at risk.     

Nutrient Uptake by ‘Alamo’ Switchgrass Used as an Energy Crop

Field studies were conducted in 2000 and 2001 to examine yields and nutrient removal by Alamo switchgrass (Panicum virgatum L.) grown at eight locations within five states in the upper southeastern USA. Plots, which had been established for >5 years as part of a larger study, were cut either once (late fall) or twice (midsummer and late fall). Plots cut once received 50 kg N per hectare per year, while twice-cut plots received 100 kg N per hectare per year. Nutrient concentrations of and nutrient removal by harvested biomass were determined. Partitioning of nutrients into leaf and stem fractions was determined at the time of the midsummer harvest in 2000. Biomass production during 2000 and 2001 averaged 15.9 Mg/ha per year across all sites and was as high as 21.7 Mg/ha per year at one site. Two cuttings plus the additional 50 kg N per hectare did not generally increase seasonal yields; and, in one quite productive location, that management caused a yield reduction. Nitrogen removal with two cuts was much higher than with a single cut due largely to the higher N content in the midsummer harvest. Over the 2 years, twice as much N was removed with the two annual cuts as with one cut. Nitrogen removal exceeded the amounts of N applied in both managements, suggesting N was being supplied via mineralization or other processes. Phosphorus removal also increased significantly with the two-cut management. Seasonal K and Ca removals were more similar between the two managements. Nitrogen and P concentrations generally declined basipetally in tillers, with older leaves and internodes having lower concentrations of both nutrients. Potassium was more uniformly distributed than N throughout the tiller components (leaf and stem). Calcium was higher in older leaf blades. Levels of soil P, K, and Ca at most locations appeared not to be limiting biomass production and were adequate for long-term productivity.     

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell‐derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non‐MP secreted factors (Sup) were isolated from serum‐free medium conditioned by human microvascular ECs (HMEC‐1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP‐containing MPs were isolated from cells transduced with CMV‐GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP‐MPs, but not free GFP. Thus, only MP‐associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP‐1, MMP‐3, CCL‐2/MCP‐1 and IL‐6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF‐κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.     

Unbinding pathways of an agonist and an antagonist from the 5-HT3 receptor

The binding sites of 5-HT3 and other Cys-loop receptors have been extensively studied, but there are no data on the entry and exit routes of ligands for these sites. Here we have used molecular dynamics simulations to predict the pathway for agonists and antagonists exiting from the 5-HT3 receptor binding site. The data suggest that the unbinding pathway follows a tunnel at the interface of two subunits, which is approximately 8 A long and terminates approximately 20 A above the membrane. The exit routes for an agonist (5-HT) and an antagonist (granisetron) were similar, with trajectories toward the membrane and outward from the ligand binding site. 5-HT appears to form many hydrogen bonds with residues in the unbinding pathway, and experiments show that mutating these residues significantly affects function. The location of the pathway is also supported by docking studies of granisetron, which show a potential binding site for granisetron on the unbinding route. We propose that leaving the binding pocket along this tunnel places the ligands close to the membrane and prevents their immediate reentry into the binding pocket. We anticipate similar exit pathways for other members of the Cys-loop receptor family.     

Dexras1 blocks receptor-mediated heterologous sensitization of adenylyl cyclase 1

Dexras1/AGS1/RasD1 is a member of the Ras superfamily of monomeric G proteins and has been suggested to disrupt receptor-G protein signaling. We examined the ability of Dexras1 to modulate dopamine D_2L receptor regulation of adenylyl cyclase (AC) type 1 in HEK293 cells. Acute D_2L receptor-mediated inhibition of A23187-stimulated AC1 activity (IC₅₀, 4.0 ± 1.4 nM; 50 ± 3% inhibition) was not altered in the presence of Dexras1 (IC₅₀, 2.4 ± 1.3 nM, 50 ± 1% inhibition); however, Dexras1 blocked acute D_2L receptor-mediated activation of ERK 1/2 by approximately 50%. Heterologous sensitization of AC1 induced by persistent activation of D_2L receptors was completely blocked by Dexras1 under basal and A23187-stimulated conditions. The block of sensitization was concentration-dependent and was not observed with a nucleotide binding-deficient Dexras1G31V mutant. Sensitization of AC1 was Gβγ-dependent as demonstrated using the C-terminus of β-adrenergic receptor kinase (βARK-ct). These data suggest that Dexras1 selectively regulates receptor-mediated Gβγ signaling pathways.