Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.

BackgroundThe ability to withstand thermal stress is considered to be of crucial importance for individual fitness and species' survival. Thus, organisms need to employ effective mechanisms to ensure survival under stressful thermal conditions, among which phenotypic plasticity is considered a particularly quick and effective one.Methodology/Principal FindingsIn a series of experiments we here investigate phenotypic adjustment in temperature stress resistance following environmental manipulations in the butterfly Bicyclus anynana. Cooler compared to warmer acclimation temperatures generally increased cold but decreased heat stress resistance and vice versa. In contrast, short-time hardening responses revealed more complex patterns, with, e.g., cold stress resistance being highest at intermediate hardening temperatures. Adult food stress had a negative effect on heat but not on cold stress resistance. Additionally, larval feeding treatment showed interactive effects with adult feeding for heat but not for cold stress resistance, indicating that nitrogenous larval resources may set an upper limit to performance under heat stress. In contrast to expectations, cold resistance slightly increased during the first eight days of adult life. Light cycle had marginal effects on temperature stress resistance only, with cold resistance tending to be higher during daytime and thus active periods.Conclusions/SignificanceOur results highlight that temperature-induced plasticity provides an effective tool to quickly and strongly modulate temperature stress resistance, and that such responses are readily reversible. However, resistance traits are not only affected by ambient temperature, but also by, e.g., food availability and age, making their measurement challenging. The latter effects are largely underexplored and deserve more future attention. Owing to their magnitude, plastic responses in thermal tolerance should be incorporated into models trying to forecast effects of global change on extant biodiversity.

RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2␣. In contrast, HIF-1␣ served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1␣ resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2␣-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2␣-dependent erythrocytosis, whereas HIF-1␣ protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia. (Blood.

Little is known about the cellular physiology of Escherichia coli at high cell densities (e.g., greater than 50 g [dry cell weight] per liter), particularly in relation to the cellular response to different growth conditions. E. coli W3100 cultures were grown under identical physical and nutritional conditions, by using a computercontrolled fermentation system which maintains the glucose concentration at 0.5 g/liter, to high cell densities at pH values of 6.0, 6.5, 7.0, and 7.5. The data suggest a relationship between the pH of the environment and the amount of acetate excreted by the organism during growth. At pH values of 6.0 and 6.5, the acetate reached a concentration of 6 g/liter, whereas at pH 7.5, the acetate reached a concentration of 12 g/liter. Furthermore, at pH values of 6.0 to 7.0, the E. coli culture undergoes a dramatic metabolic switch in which oxygen and glucose consumption and CO2 evolution all temporarily decrease by 50 to 80%, with a concomitant initiation of acetate utilization. After a 30-min pause in which approximately 50% of the available acetate is consumed, the culture recovers and resumes consuming glucose and oxygen and producing acetate and CO2 at preswitch levels. During the switch period, the specific activity of isocitrate lyase typically increases approximately fourfold.It is well known that during rapid aerobic growth, strains of Escherichia coli produce acetic acid as a by-product (7, 9-14, 17, 19), although the amount of acetate produced is strain dependent (17). Luli and Strohl (17) found that more than a threefold difference in acetate concentrations may occur between different strains grown in batch fermentations under identical conditions. The growth rate of the culture and the composition of the medium also influence the amount of acetate produced during growth. In chemostat experiments, acetate is not produced until the growth rate reaches a threshold value that is dependent upon the type of growth medium (21). E. coli cultures will produce acetate at a lower growth rate when grown in a nutrient-rich medium than in a defined medium (9, 21), and the specific acetate production rate also changes with the growth rate (9).Acetate is produced when the uptake of the primary carbon source is greater than its conversion to biomass and CO2 (10). The rate-limiting aspect of metabolism contributing to acetic acid formation by E. coli has been attributed to the electron transport system (7), the tricarboxylic acid cycle (10), or a combination of both (19). In a batch culture growing on glucose, approximately 15% of the carbon input is typically excreted as acetate (10).The presence of acetate in the growth medium can influence the physiology of the cell in many ways. Elevated acetate concentrations inhibit the growth rate of the culture, the effect of which may be more pronounced in complex medium than in defined medium (23). It has been postulated that the protonated form of acetate can cross the cytoplasmic membrane and reduce the proton motive force by decreasing ApH (7, 17), a...