Proteasome control of [URE3] prion propagation by degradation of anti-prion proteins Cur1 and Btn2 in Saccharomyces cerevisiae

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion—curing effects, each mutation affecting proteasomes elevates the cellular concentration of the antiprion proteins Btn2 and Cur1. Of > 4600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion—curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.

Extended erythrocyte and reticulocyte parameters in the diagnosis of iron deficiency in hemodialysis patients

Objective: Iron deficiency (ID) is a frequent complication in end stage renal insufficiency. These patients have to be diagnosed and treated to reduce the prevalence of anemia. Functional iron deficiency (FID) is a situation that can disrupt biochemical iron tests and mask an eventual association with ID. In this study, we tried to prove the ability of extended parameters of red cells and reticulocytes to diagnose ID without being influenced by FID. Design and methods: 164 chronic hemodialysis patients (CHP) in end stage renal disease were enrolled. Research parameters of red cells and reticulocytes determined on ADVIA 2120i were studied in the diagnosis of ID associated or not with chronic inflammation. Results: Parameters such as corpuscular hemoglobin of mature red cells (CHm), corpuscular hemoglobin of reticulocytes (CHr), cellular concentration of hemoglobin in mature red cells (CHCMm), cellular concentration of hemoglobin in reticulocytes (CHCMr) and percentage of microcytic and hypochromic red cells (HYMI) showed a high sensitivity to diagnose ID. However, the distinction of combined iron deficiency (CID) from other entities was not possible with all parameters. In chronic inflammatory states, the decrease of CHm, CHCMm and CHCMr with the rise of percentage hypochromic mature red cells (HYPOm) and reticulocytes (HYPOr) is in favor of CID. So, determination of inflammatory state is needed to complete research parameters of blood count in CHP. Conclusion: Extended erythrocyte and reticulocyte parameters can be useful to check iron status in CHP.

Comparison of growth and toxicity responses between non-toxic and toxic strains of Prorocentrum hoffmannianum

We aimed to study the growth and toxicity responses of non-toxic (CCMP683) and toxic (CCMP2804) strains of Prorocentrum hoffmannianum under various nitrate and phosphate concentrations. The 2 strains were cultured in L1-Si medium with standard, depleted or 10-fold repleted nitrate or phosphate. CCMP683 cultured in standard L1-Si medium exhibited delayed growth. Nitrate or phosphate depletion decreased the cell density of both strains. Repletion of nitrate slightly increased the cell density of both strains. Repletion of phosphate also slightly increased the cell density of CCMP2804 but surprisingly decreased the cell density of CCMP683. Okadaic acid (OA) and its derivatives were not detected in CCMP683. OA was detected only in CCMP2804. Depletion of nitrate or phosphate increased the cellular concentration of OA, and repletion of nitrate or phosphate had no effect on the cellular concentration of OA. Correlation analysis indicated that the cellular concentration of OA was negatively correlated with cell density. Differences in the growth response to phosphate repletion and in the ability to produce OA suggest that the 2 strains may be good candidates for comparative studies related to phosphate metabolism and OA toxicity.

Hemoglobin F or: How I Learned to Stop Wondering and Love the Flow Cytometer

Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. It is a hemoglobinopathy that leads to red blood cell (RBC) sickling and a broad range of disease complications including vaso-occlusive crisis, acute chest syndrome, and retinopathy. Hydroxyurea, a drug used to treat SCD, is known to increase expression of hemoglobin F (HbF), a type of hemoglobin normally expressed in infancy; HbF levels between 10% and 20% are associated with decreased vaso-occlusive episodes and improved survival. Hereditary persistent hemoglobin F (HPHF), a typically asymptomatic hemoglobinopathy associated with sustained hemoglobin F (HbF) expression into adulthood (HbF >10%), in combination with SCD is associated with decreased complications. Laboratories typically determine the HbF level via high-performance liquid chromatography (HPLC). HbF levels approaching 30% on HPLC are thought to be protective against SCD complications. However, HbF may be found within a majority or minority of RBCs, pancellular (deletional HPHF) or heterocellular distribution (nondeletional HPHF), respectively. Additionally, the quantity of HbF within cells can range from low (<10 picograms/cell) to high (>35 picograms). We sought to determine the quantity and distribution of HbF required to protect against sickle cell disease symptoms both via traditional HPLC as well as flow cytometry. This retrospective study was conducted at a large academic medical center over a period of 2 months (January-February 2019). We collected blood from sickle cell patients that had a detectable HbF level on hemoglobin electrophoresis. We then stained RBCs from 16 of the patients for HbF and performed flow cytometry to examine the HbF distribution. We calculated the cellular concentration of HbF within each HbF+ cell using the formula (MHC × %HbF)/%F-cells. We performed a chart review to determine the native hemoglobin type, exposure to hydroxyurea, and clinical symptoms of sickle cell disease. We identified four patients over the age of 20 with HbS/HPHP and no exposure to hydroxyurea. Two of these patients experienced no sickle cell disease complications; the protected patients had heterocellular distribution of HbF, but had a high concentration of HbF per HbF+ cell (>35 picograms/cell). Notably, these asymptomatic patients both had HbF level by HPLC less than 30. One of the symptomatic HbS/HPHF patients had heterocellular expression of HbF with low cellular concentration (28 picograms/cell) while the other patient had pancellular HbF expression with very low cellular concentration (6.4 picograms/cell). Our study demonstrates that HPHF alone does not prevent sickle cell disease complications. Our study highlights the importance of quantifying the cellular concentration of HbF, which can provide useful information beyond that of HPLC. In addition, our study raises the potential of the clinical use of hydroxyurea in patients with sickle cell disease even in the presence of HPHF.

Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift inEscherichia coli

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT inEscherichia coliand related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented inE. coli. In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response inE. coli.

EVALUACION DE DIFERENTES DENSIDADES DE LA MICROALGA NANNOCHLOROPSIS OCULATA COMO ALIMENTO PARA ARTEMIA

Laboratory experiments were carried out to determine the possibility of using the microalgae Nannochloropsis oculata as a food source for brine shirmp. Among the range concentrations studied (3 x 105 to 14 x 105 cells/ml), 12 x 105 cells/ml offered the best results of survival and growth rate and it is recommended as the most effective. From correlation matrix, high values (r > 0.90; a = 0.05) were obtained for the relation of microalgae concentration and survival, growth rate and the variation of average lenght. The minimum cellular concentration of microalgae which is necessary for brine shrimp culture is 5.6 x 105 cells/ml.