Non-transferrin bound iron,cytokine activation and intracellular reactive oxygen species generation in hemodialysis patients receiving intravenous iron dextran or iron sucrose

Intravenous (IV) iron supplementation is widely used to support erythropoeisis in hemodialysis patients. IV iron products are associated with oxidative stress that has been measured principally by circulating biomarkers such as products of lipid peroxidation. The pro-oxidant effects of IV iron are presumed to be due at least in part, by free or non-transferrin bound iron (NTBI). However, the effects of IV iron on intracellular redox status and downstream effectors is not known. This prospective, crossover study compared cytokine activation, reactive oxygen species generation and oxidative stress after single IV doses of iron sucrose and iron dextran. This was a prospective, open-label, crossover study. Ten patients with end-stage renal disease (ESRD) on hemodialysis and four age and sex-matched healthy were assigned to receive 100 mg of each IV iron product over 5 min in random sequence with a 2 week washout between products. Subjects were fasted and fed a low iron diet in the General Clinical Research Center at the University of New Mexico. Serum and plasma samples for IL-1, IL-6, TNF-α and IL-10 and NTBI were obtained at baseline, 60 and 240 min after iron infusion. Peripheral blood mononuclear cells (PBMC) were isolated at the same time points and stained with fluorescent probes to identify intracellular reactive oxygen species and mitochondrial membrane potential (Δψm) by flow cytometry. Lipid peroxidation was assessed by plasma F₂ isoprostane concentration. Mean ± SEM maximum serum NTBI values were significantly higher among patients receiving IS compared to ID (2.59 ± 0.31 and 1.0 ± 0.36 μM, respectively, P = 0.005 IS vs. ID) Mean ± SEM NTBI area under the serum concentration–time curve (AUC) was 3-fold higher after IS versus ID (202 ± 53 vs. 74 ± 23 μM*min/l, P = 0.04) in ESRD patients, indicating increased exposure to NTBI. IV iron administration was associated with increased pro-inflammatory cytokines. Serum IL-6 concentrations increased most profoundly, with a 2.6 and 2.1 fold increase from baseline in ESRD patients given IS and ID, respectively (P < 0.05 compared to baseline). In healthy controls, serum IL-6 was undetectable at baseline and after IV iron administration. Most ESRD patients had increased intracellular ROS generation, however, there was no difference between ID and IS. Only one healthy control had increased ROS generation post IV iron. All healthy controls experienced a loss of Δψm (100% with IS and 50% with ID). ESRD patients also had loss of Δψm with a nadir at 240 min. IS administration was associated with higher maximum serum NTBI concentrations compared to ID, however, the both compounds produced similar ROS generation and cytokine activation that was more pronounced among ESRD patients. The effect of IV iron-induced ROS production on pivotal signaling pathways needs to be explored.     

Mutational and Structural Analysis of the Tandem Zinc Finger Domain of Tristetraprolin

Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX₈CX₅CX₃H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3′-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3′-UTR. Within the CX₈C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX₅C or CX₃H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.     

An agent based approach to the potential for rebound resulting from evolution of residential lighting technologies

Purpose

More energy efficient lighting options, such as compact fluorescent bulbs and light emitting diodes are predicted to significantly reduce the amount of energy used for lighting. Such forecasts are predicated on the assumption of light saturation and do not take into account the potential for economic rebound. The potential of the rebound effect to reduce or negate predicted energy savings is explored here.

Methods

This work uses an agent-based model with a cellular automata approach to study the impact of rebound on the consumption of residential light and associated energy use, using three lighting technologies, and a time span from 2012 to 2030. Agents, representative of households, select between three lighting options using a multiplicative utility function and a probabilistic choice mechanism. Agents then decide whether to consume more light and potentially more energy based on the lighting technology selected and personal preferences. The agents are heterogeneous in nature, consisting of seven typologies, with their characteristics informed through survey data.

Results and discussion

The results of the model indicate that although the consumption of light may increase, overall changes in the consumption of energy compared to 2012 levels will be minor. If the consumption of light is held steady, assuming saturation, then there is the potential for the adoption of energy-efficient lighting to result in significant energy savings. However, if the rebound effect occurs, there will be a decrease in the consumption of energy for lighting as consumers adopt more energy efficient options. Overtime as the consumption of light continues to increase, those savings will largely be eroded.

Conclusions

This study suggests that the adoption of energy-efficient lighting in itself will not reduce the overall consumption of energy for lighting on a long-term scale although it may be successful in doing so in the short-term. The rebound effect will greatly reduce the projected energy savings from more efficient lighting technologies, with potential for direct rebound to exceed 100 %. In order for the quantity of energy utilized in residential lighting to decrease, solutions beyond that of efficiency gains must be considered.     

The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane

Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community.Chlamydomonas reinhardtii (C. reinhardtii)¹ is the most intensively studied and well-developed microalgal model species for investigation of a wide-range of processes ranging from basic development through understanding triacylglycerol production. C. reinhardtii is easy to culture, grows quickly, and is tolerant to varying growth conditions. Additionally, the genome of C. reinhardtii is sequenced (1) and C. reinhardtii is easily engineered at the genetic level (2), thus making it an attractive model system for investigation of a wide range of underlying biology processes, including photosynthesis, cell motility, and phototaxis, cell-wall biogenesis, and other fundamental cellular processes (3).Advances in proteomic technologies permit ever increasing breadth and depth for interrogation of protein level dynamics, and the definitive role of phosphorylation in affecting a protein''s function, activity, localization, stability, and ligand/protein interactions is well understood (4). However, compared with Arabidopsis and other plant species (5), the C. reinhardtii phosphoproteome data set is still in nascent assembly. In a series of studies, researchers investigated the effects of environmental changes on 43 phosphopeptides among thylakoid membrane-associated proteins (6–8), analysis of which provides evidence for a thylakoid protein kinase cascade. Wagner et al. (9) observed 83 phosphopeptides associated with the eyespot apparatus, including several kinases and phosphatases implicated in phosphorylation-based signaling in the eyespot. In a study of C. reinhardtii flagella, Pan et al. (10) observed 1296 spectral counts of phosphopeptides corresponding to 224 phosphoproteins involved with motility and assembly. In a similar study, Boesger et al. (11) observed 141 phosphopeptides corresponding to 32 proteins. Using whole cells, Wagner et al. (12) observed 360 phosphopeptides corresponding to 328 proteins, including several flagellar kinases, which indicates the importance of phosphorylation-based signaling for motility and assembly.Despite the importance of phosphorylation-based signaling underlying C. reinhardtii biological processes, characterization of the cellular pool of phosphopeptides has been limited. Although additional dimensions of separation that are orthogonal to online reversed-phase are routinely used in order to probe phosphopeptide species of low-abundance, this has not been implemented for probing the C. reinhardtii phosphoproteome. Hydrophilic-interaction liquid chromatography improves phosphopeptide separation and detection (13) and is more orthogonal than strong-cation exchange compared with online reversed-phase chromatography (14). Additionally, to complement the increased resolution of phosphopeptides afforded by a first-dimension separation, enrichment strategies based on the affinity of a phosphate group to a metal ion or metal oxide can further increase coverage. Currently, a single immobilized metal affinity chromatography (IMAC) scheme is the most popular choice for phosphopeptide studies using C. reinhardtii. However, conventional insoluble TiO₂ beads recover more phosphopeptides than traditional IMAC (15). Additionally, PolyMAC (polymer-based metal ion affinity capture) is a polymer-based improved analog of IMAC that uses TiO₂-functionalized soluble nanopolymers to chelate phosphopeptides in a homogeneous aqueous environment (15). Thus, use of complementary enrichment schemes based on TiO₂ and PolyMAC can yield more comprehensive results compared with a single strategy.In this study, complementary approaches using TiO₂/PolyMAC enrichment and hydrophilic-interaction liquid chromatography (HILIC) chromatography were employed to explore the C. reinhardtii phosphoproteome in significant depth. We report the detection of 4588 nonredundant phosphoproteins from 18,160 unique phosphopeptides at <1% false discovery rate. Among these peptides, we report 15,862 unique phosphosites identified with ≥95% localization probability. Nearly all reported sites are novel. Our data show many key biological pathways, including photosynthesis, chlorophyll biosynthesis, carbon assimilation, protein metabolism, and flagella assembly and motility are comprised of multiple phosphoproteins. These data provide a framework for garnering novel mechanistic insights into understanding a variety of cellular/signaling processes.     

Dysregulated Estrogen Receptor Signaling in the Hypothalamic-Pituitary-Ovarian Axis Leads to Ovarian Epithelial Tumorigenesis in Mice

The etiology of ovarian epithelial cancer is poorly understood, mainly due to the lack of an appropriate experimental model for studying the onset and progression of this disease. We have created a mutant mouse model in which aberrant estrogen receptor alpha (ERα) signaling in the hypothalamic-pituitary-ovarian axis leads to ovarian epithelial tumorigenesis. In these mice, termed ERα^d/d, the ERα gene was conditionally deleted in the anterior pituitary, but remained intact in the hypothalamus and the ovary. The loss of negative-feedback regulation by estrogen (E) at the level of the pituitary led to increased production of luteinizing hormone (LH) by this tissue. Hyperstimulation of the ovarian cells by LH resulted in elevated steroidogenesis, producing high circulating levels of steroid hormones, including E. The ERα^d/d mice exhibited formation of palpable ovarian epithelial tumors starting at 5 months of age with 100% penetrance. By 15 months of age, 80% of ERα^d/d mice die. Besides proliferating epithelial cells, these tumors also contained an expanded population of luteinized stromal cells, which acquire the ability to express P450 aromatase and synthesize E locally. In response to the elevated levels of E, the ERα signaling was accentuated in the ovarian epithelial cells of ERα^d/d mice, triggering increased ERα-dependent gene expression, abnormal cell proliferation, and tumorigenesis. Consistent with these findings, treatment of ERα^d/d mice with letrozole, an aromatase inhibitor, markedly reduced circulating E and ovarian tumor volume. We have, therefore, developed a unique animal model, which serves as a useful tool for exploring the involvement of E-dependent signaling pathways in ovarian epithelial tumorigenesis.     

Non-Invasive Prenatal Chromosomal Aneuploidy Testing - Clinical Experience: 100,000 Clinical Samples

Objective

As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT.

Study Design

The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA–licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting.

Results

NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result.

Conclusion

NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.     

Characterization of the Inflammatory Microenvironment and Identification of Potential Therapeutic Targets in Wilms Tumors

The role of inflammation in cancer has been reported in various adult malignant neoplasms. By contrast, its role in pediatric tumors has not been as well studied. In this study, we have identified and characterized the infiltration of various inflammatory immune cells as well as inflammatory markers in Wilms tumor (WT), the most common renal malignancy in children. Formalin-fixed paraffin-embedded blocks from tumors and autologous normal kidneys were immunostained for inflammatory immune cells (T cells, B cells, macrophages, neutrophils, and mast cells) and inflammatory markers such as cyclooxygenase-2 (COX-2), hypoxia-inducible factor 1α, phosphorylated STAT3, phosphorylated extracellular signal–related kinases 1 and 2, inducible nitric oxide synthase, nitrotyrosine, and vascular endothelial growth factor expression. Overall, we found that there was predominant infiltration of tumor-associated macrophages in the tumor stroma where COX-2 was robustly expressed. The other tumor-associated inflammatory markers were also mostly localized to tumor stroma. Hence, we speculate that COX-2–mediated inflammatory microenvironment may be important in WT growth and potential therapies targeting this pathway may be beneficial and should be tested in clinical settings for the treatment of WTs in children.     

Life histories of closely related amphidromous and non‐migratory fish species: a trade‐off between egg size and fecundity

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