The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4⁺ CD8⁺ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3^−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

Four Weeks of IV Iron Supplementation Reduces Perceived Fatigue and Mood Disturbance in Distance Runners

Purpose

To determine the effect of intravenous iron supplementation on performance, fatigue and overall mood in runners without clinical iron deficiency.

Methods

Fourteen distance runners with serum ferritin 30–100 µg·L⁻¹ were randomly assigned to receive three blinded injections of intravenous ferric-carboxymaltose (2 ml, 100 mg, IRON) or normal saline (PLACEBO) over four weeks (weeks 0, 2, 4). Athletes performed a 3,000 m time trial and 10×400 m monitored training session on consecutive days at week 0 and again following each injection. Hemoglobin mass (Hbmass) was assessed via carbon monoxide rebreathing at weeks 0 and 6. Fatigue and mood were determined bi-weekly until week 6 via Total Fatigue Score (TFS) and Total Mood Disturbance (TMD) using the Brief Fatigue Inventory and Brunel Mood Scale. Data were analyzed using magnitude-based inferences, based on the unequal variances t-statistic and Cohen''s Effect sizes (ES).

Results

Serum ferritin increased in IRON only (Week 0: 62.8±21.9, Week 4: 128.1±46.6 µg·L⁻¹; p = 0.002) and remained elevated two weeks after the final injection (127.0±66.3 µg·L⁻¹, p = 0.01), without significant changes in Hbmass. Supplementation had a moderate effect on TMD of IRON (ES -0.77) with scores at week 6 lower than PLACEBO (ES -1.58, p = 0.02). Similarly, at week 6, TFS was significantly improved in IRON vs. PLACEBO (ES –1.54, p = 0.05). There were no significant improvements in 3,000 m time in either group (Week 0 vs. Week 4; Iron: 625.6±55.5 s vs. 625.4±52.7 s; PLACEBO: 624.8±47.2 s vs. 639.1±59.7 s); but IRON reduced their average time for the 10×400 m training session at week 2 (Week 0: 78.0±6.6 s, Week 2: 77.2±6.3; ES–0.20, p = 0.004).

Conclusion

During 6 weeks of training, intravenous iron supplementation improved perceived fatigue and mood of trained athletes with no clinical iron deficiency, without concurrent improvements in oxygen transport capacity or performance.     

Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant benefits of therapy

Chelation therapy is thought to not only remove contaminating metals but also to decrease free radical production. EDTA chelation therapy, containing high doses of vitamin C as an antioxidant, is often used in the treatment of diseases such as diabetes and cardiovascular diseases but the effectiveness of this treatment may be variable and its efficacy has not been demonstrated conclusively. The objective of this work was to determine if the vitamin C added to standard chelation therapy cocktails was prooxidant. We administered a standard EDTA cocktail solution with or without 5 g of sodium ascorbate. One hour following the standard chelation therapy, there were highly significant prooxidant effects on lipids, proteins, and DNA associated with decreased activities of RBC glutathione peroxidase and superoxide dismutase while in the absence of sodium ascorbate, there were no acute signs of oxidative damage. After 16 sessions of standard chelation therapy, the acute prooxidant effects of vitamin C remained, but, even in the absence of nutrient supplements, there were beneficial long-term antioxidant effects of chelation therapy and plasma peroxide levels decreased. In conclusion, multiple sessions of EDTA chelation therapy protect lipids against oxidative damage. However, standard high amounts of vitamin C added to EDTA chelation solutions also display short term prooxidant effects. The added benefits of lower levels of vitamin C in chelation therapy need to be documented.     

In vivo assessment and potential diagnosis of xenobiotics that perturb the thyroid pathway: Proteomic analysis of Xenopus laevis brain tissue following exposure to model T4 inhibitors

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ®) and LC–MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.     

Using linkage models to explore skull kinematic diversity and functional convergence in arthrodire placoderms

Biomechanical models offer a powerful set of tools for quantifying the diversity of function across fossil taxa. A computer‐based four‐bar linkage model previously developed to describe the potential feeding kinematics of Dunkleosteus terrelli is applied here to several other arthrodire placoderm taxa from different lineages. Arthrodire placoderms are a group of basal gnathostomes showing one of the earliest diversifications of jaw structures. The linkage model allows biomechanical variation to be compared across taxa, identify trends in skull morphology among arthrodires that potentially influence function and explore the role of linkage systems in the early evolution of jaw structures. The linkage model calculates various kinematic metrics including gape angle, effective mechanical advantage, and kinematic transmission coefficients. Results indicate that the arthrodire feeding system may be more diverse and complex than previously thought. A range of potential kinematic profiles among arthrodire taxa illustrate a diversity of feeding function comparable with modern teleost fishes. Previous estimates of bite force in Dunkleosteus are revised based on new morphological data. High levels of kinematic transmission among arthrodires suggest the potential for rapid gape expansion and possible suction feeding. Morphological comparisons indicate that there were several morphological solutions for obtaining these fast kinematics, which allowed different taxa to achieve similar kinematic profiles while varying other aspects of the feeding apparatus. Mapping of key morphological components of the linkage system on a general placoderm phylogeny illustrates the potential importance of four‐bar systems to the early evolution of jaw structures. J. Morphol. 271:990–1005, 2010. © 2010 Wiley‐Liss, Inc.     

Placental specializations of the mountain spiny lizard Sceloporus jarrovi

The lizard Sceloporus jarrovi (Phrynosomatidae) is one of the most widely studied viviparous reptiles of North America. Past research has assumed that placentation in this species is relatively simple and functions mainly in gas exchange. Our examination of the late stage placenta via transmission electron microscopy reveals that S. jarrovi has a unique combination of placental characteristics, with unusual specializations for secretion and absorption. In the chorioallantoic placenta, chorionic and uterine tissues are directly apposed through eggshell loss, and their epithelia are greatly attenuated, enhancing gas exchange; this placenta shows evidence of both nutrient transfer and endocrine function. Contrary to past inferences, a yolk sac placenta forms from the avascular omphalopleure and persists through the end of gestation. The uterine epithelium is enlarged and secretory, and the fetal omphalopleure shows branching absorptive channels and other specializations for uptake. Elsewhere, the omphalopleure develops elongated folds that protrude into a coagulum of degenerating shell membrane and other organic material. Uterine tissue in this region shows specializations for absorption. Placental features in S. jarrovi have unexpected functional implications, and challenge assumptions that specializations for nutrient transfer are confined to matrotrophic species. J. Morphol. 271:1153–1175, 2010. © 2010 Wiley‐Liss, Inc.     

Growth and Maximum Size of Tiger Sharks (Galeocerdo cuvier) in Hawaii

Tiger sharks (Galecerdo cuvier) are apex predators characterized by their broad diet, large size and rapid growth. Tiger shark maximum size is typically between 380 & 450 cm Total Length (TL), with a few individuals reaching 550 cm TL, but the maximum size of tiger sharks in Hawaii waters remains uncertain. A previous study suggested tiger sharks grow rather slowly in Hawaii compared to other regions, but this may have been an artifact of the method used to estimate growth (unvalidated vertebral ring counts) compounded by small sample size and narrow size range. Since 1993, the University of Hawaii has conducted a research program aimed at elucidating tiger shark biology, and to date 420 tiger sharks have been tagged and 50 recaptured. All recaptures were from Hawaii except a single shark recaptured off Isla Jacques Cousteau (24°13′17″N 109°52′14″W), in the southern Gulf of California (minimum distance between tag and recapture sites  =  approximately 5,000 km), after 366 days at liberty (DAL). We used these empirical mark-recapture data to estimate growth rates and maximum size for tiger sharks in Hawaii. We found that tiger sharks in Hawaii grow twice as fast as previously thought, on average reaching 340 cm TL by age 5, and attaining a maximum size of 403 cm TL. Our model indicates the fastest growing individuals attain 400 cm TL by age 5, and the largest reach a maximum size of 444 cm TL. The largest shark captured during our study was 464 cm TL but individuals >450 cm TL were extremely rare (0.005% of sharks captured). We conclude that tiger shark growth rates and maximum sizes in Hawaii are generally consistent with those in other regions, and hypothesize that a broad diet may help them to achieve this rapid growth by maximizing prey consumption rates.     

Microbial Growth and Carbon Use Efficiency in the Rhizosphere and Root-Free Soil

Plant-microbial interactions alter C and N balance in the rhizosphere and affect the microbial carbon use efficiency (CUE)–the fundamental characteristic of microbial metabolism. Estimation of CUE in microbial hotspots with high dynamics of activity and changes of microbial physiological state from dormancy to activity is a challenge in soil microbiology. We analyzed respiratory activity, microbial DNA content and CUE by manipulation the C and nutrients availability in the soil under Beta vulgaris. All measurements were done in root-free and rhizosphere soil under steady-state conditions and during microbial growth induced by addition of glucose. Microorganisms in the rhizosphere and root-free soil differed in their CUE dynamics due to varying time delays between respiration burst and DNA increase. Constant CUE in an exponentially-growing microbial community in rhizosphere demonstrated the balanced growth. In contrast, the CUE in the root-free soil increased more than three times at the end of exponential growth and was 1.5 times higher than in the rhizosphere. Plants alter the dynamics of microbial CUE by balancing the catabolic and anabolic processes, which were decoupled in the root-free soil. The effects of N and C availability on CUE in rhizosphere and root-free soil are discussed.