Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.     

Donor APCs are required for maximal GVHD but not for GVL

Graft-versus-host disease (GVHD) is a major source of morbidity in allogenic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD in a mouse model across only minor histocompatibility antigens (minor H antigens). However, these studies did not address the function of donor-derived APCs after GVHD is initiated. Here we show that GVHD develops in recipients of donor major histocompatibility complex class I-deficient (MHC I(-)) bone marrow. Thus, after initial priming, CD8 cells caused GVHD without a further requirement for hematopoietic APCs, indicating that host APCs are necessary and sufficient for GHVD. Nonetheless, GVHD was less severe in recipients of MHC I(-) bone marrow. Therefore, once initiated, GVHD is intensified by donor-derived cells, most probably donor APCs cross-priming alloreactive CD8 cells. Nevertheless, donor APCs were not required for CD8-mediated graft-versus-leukemia (GVL) against a mouse model of chronic-phase chronic myelogenous leukemia. These studies identify donor APCs as a new target for treating GVHD, which may preserve GVL.     

Automatic Segmentation of Eight Tissue Classes in Neonatal Brain MRI

Purpose

Volumetric measurements of neonatal brain tissues may be used as a biomarker for later neurodevelopmental outcome. We propose an automatic method for probabilistic brain segmentation in neonatal MRIs.

Materials and Methods

In an IRB-approved study axial T1- and T2-weighted MR images were acquired at term-equivalent age for a preterm cohort of 108 neonates. A method for automatic probabilistic segmentation of the images into eight cerebral tissue classes was developed: cortical and central grey matter, unmyelinated and myelinated white matter, cerebrospinal fluid in the ventricles and in the extra cerebral space, brainstem and cerebellum. Segmentation is based on supervised pixel classification using intensity values and spatial positions of the image voxels. The method was trained and evaluated using leave-one-out experiments on seven images, for which an expert had set a reference standard manually. Subsequently, the method was applied to the remaining 101 scans, and the resulting segmentations were evaluated visually by three experts. Finally, volumes of the eight segmented tissue classes were determined for each patient.

Results

The Dice similarity coefficients of the segmented tissue classes, except myelinated white matter, ranged from 0.75 to 0.92. Myelinated white matter was difficult to segment and the achieved Dice coefficient was 0.47. Visual analysis of the results demonstrated accurate segmentations of the eight tissue classes. The probabilistic segmentation method produced volumes that compared favorably with the reference standard.

Conclusion

The proposed method provides accurate segmentation of neonatal brain MR images into all given tissue classes, except myelinated white matter. This is the one of the first methods that distinguishes cerebrospinal fluid in the ventricles from cerebrospinal fluid in the extracerebral space. This method might be helpful in predicting neurodevelopmental outcome and useful for evaluating neuroprotective clinical trials in neonates.     

Detection of viral hemorrhagic septicemia in round gobies in New York State (USA) waters of Lake Ontario and the St. Lawrence River

In May 2006 a large mortality of several thousand round gobies Neogobius melanostomus (Pallas, 1814) occurred in New York waters of the St. Lawrence River and Lake Ontario. Necropsies of sampled fish from these areas showed pallor of the liver and gills, and hemorrhagic areas in many organs. Histopathologic examination of affected tissues revealed areas of necrosis and hemorrhage. Inoculations of fathead minnow Pimephales promelas (Rafinesque, 1820) cell cultures with dilutions of tissue samples from the necropsied gobies produced a cytopathic effect within 5 d post-inoculation. Samples of cell culture supernatant were tested using RT-PCR and confirmed the presence of viral hemorrhagic septicemia virus (VHSV). Sequence analysis of the VHSV isolate resulted in its assignment to the type-IVb subgroup. The detection of VHSV in a relatively recent invasive fish species in the Great Lakes and the potential impact of VHSV on the ecology and economy of the area will require further investigation and careful management considerations.     

Spatial analysis of a hydrocarbon waste-remediating landfarm demonstrates influence of management practices on bacterial and fungal community structure

Cultivation of dedicated soil plots called ‘landfarms' is an effective technology for bioremediation of hydrocarbon waste generated by various industrial practices. To understand the influence of soil conditions on landfarm microbial communities, analysis of bacterial and fungal community structure using next-generation sequencing at different sections and depths was performed across a hydrocarbon-waste landfarm in Regina, Saskatchewan, Canada. While a core set of hydrocarbon-associated bacterial and fungal taxa are present throughout the landfarm, unique bacterial and fungal operational taxonomic units are differentially abundant at sections within the landfarm, which correlate with differences in soil physiochemical properties and management practices. Increased frequency of waste application resulted in strong positive correlations between bacterial community assemblages and elevated amounts of oil, grease and F3 – F4 hydrocarbon fractions. In areas of standing water and lower application of hydrocarbon, microbial community structure correlated with soil pH, trace nutrients and metals. Overall, diversity and structure of bacterial communities remain relatively stable across the landfarm, while in contrast, fungal community structure appears more responsive to soil oxygen conditions. Results are consistent with the hypothesis that years of bioremediation activity have shaped microbial communities; however, several management practices can be undertaken to increase efficiency of remediation, including the removal of standing water and soil tilling across the landfarm.     

Maternal Condition but Not Corticosterone Is Linked to Offspring Sex Ratio in a Passerine Bird

There is evidence of offspring sex ratio adjustment in a range of species, but the potential mechanisms remain largely unknown. Elevated maternal corticosterone (CORT) is associated with factors that can favour brood sex ratio adjustment, such as reduced maternal condition, food availability and partner attractiveness. Therefore, the steroid hormone has been suggested to play a key role in sex ratio manipulation. However, despite correlative and causal evidence CORT is linked to sex ratio manipulation in some avian species, the timing of adjustment varies between studies. Consequently, whether CORT is consistently involved in sex-ratio adjustment, and how the hormone acts as a mechanism for this adjustment remains unclear. Here we measured maternal baseline CORT and body condition in free-living blue tits (Cyanistes caeruleus) over three years and related these factors to brood sex ratio and nestling quality. In addition, a non-invasive technique was employed to experimentally elevate maternal CORT during egg laying, and its effects upon sex ratio and nestling quality were measured. We found that maternal CORT was not correlated with brood sex ratio, but mothers with elevated CORT fledged lighter offspring. Also, experimental elevation of maternal CORT did not influence brood sex ratio or nestling quality. In one year, mothers in superior body condition produced male biased broods, and maternal condition was positively correlated with both nestling mass and growth rate in all years. Unlike previous studies maternal condition was not correlated with maternal CORT. This study provides evidence that maternal condition is linked to brood sex ratio manipulation in blue tits. However, maternal baseline CORT may not be the mechanistic link between the maternal condition and sex ratio adjustment. Overall, this study serves to highlight the complexity of sex ratio adjustment in birds and the difficulties associated with identifying sex biasing mechanisms.     

Assessing the effects of repeated handling on the physiology and condition of semi‐precocial nestlings

Repeated exposure to elevated levels of glucocorticoids during development can have long‐term detrimental effects on survival and fitness, potentially associated with increased telomere attrition. Nestling birds are regularly handled for ecological research, yet few authors have considered the potential for handling‐induced stress to influence hormonally mediated phenotypic development or bias interpretations of subsequent focal measurements. We experimentally manipulated the handling experience of the semi‐precocial nestlings of European Storm Petrel Hydrobates pelagicus to simulate handling in a typical field study and examined cumulative effects on physiology and condition in late postnatal development. Neither baseline corticosterone (the primary glucocorticoid in birds), telomere length nor body condition varied with the number of handling episodes. The absence of a response could be explained if Storm Petrels did not perceive handling to be stressful or if there is dissociation of the hypothalamic–pituitary–adrenal axis from stressful stimuli in early life. Eliciting a response to a stressor may be maladaptive for cavity‐dwelling young that are unable to escape or defend themselves. Furthermore, avoiding elevated overall glucocorticoid exposure may be particularly important in a long‐lived species, in which accelerated early‐life telomere erosion could impact negatively upon longevity. We propose that the level of colony‐wide disturbance induced by investigator handling of young could be important in underlining species‐specific responses. Storm Petrel nestlings appear unresponsive to investigator handling within the limits of handling in a typical field study and handling at this level should not bias physiological and morphological measurements.     

X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase

Previous studies of ferrous wild-type phenylalanine hydroxylase, [Fe(2+)]PAH(T)[], have shown the active site to be a six-coordinate distorted octahedral site. After the substrate and cofactor bind to the enzyme ([Fe(2+)]PAH(R)[L-Phe,5-deaza-6-MPH(4)]), the active site converts to a five-coordinate square pyramidal structure in which the identity of the missing ligand had not been previously determined. X-ray absorption spectroscopy (XAS) at the Fe K-edge further supports this coordination number change with the binding of both cosubstrates to the enzyme, and determines this to be due to the loss of a water ligand.     

TGF-β1 as possible link between loss of bone mineral density and chronic inflammation

Background

The TGF family plays a key role in bone homeostasis. Systemic or topic application of proteins of this family apparently positively affects bone healing in vivo. However, patients with chronic inflammation, having increased TGF-β₁ serum-levels, often show reduced bone mineral content and disturbed bone healing. Therefore, we wanted to identify intracellular mechanisms induced by chronic presence of TGF-β₁ and their possible role in bone homeostasis in primary human osteoblasts.

Methodology/Principal Findings

Osteoblasts were isolated from femur heads of patients undergoing total hip replacement. Adenoviral reporter assays showed that in primary human osteoblasts TGF-β₁ mediates its signal via Smad2/3 and not Smad1/5/8. It induces proliferation as an intermediate response but decreases AP-activity and inorganic matrix production as a late response. In addition, expression levels of osteoblastic markers were strongly regulated (AP↓; Osteocalcin↓; Osteopontin↑; MGP↓; BMP 2↓; BSP2↓; OSF2↓; Osteoprotegerin↓; RANKL↑) towards an osteoclast recruiting phenotype. All effects were blocked by inhibition of Smad2/3 signaling with the Alk5-Inhibitor (SB431542). Interestingly, a rescue experiment showed that reduced AP-activities did not recover to base line levels, even 8 days after stopping the TGF-β₁ application.

Conclusions/Significance

In spite of the initial positive effects on cell proliferation, it is questionable if continuous Smad2/3 phosphorylation is beneficial for bone healing, because decreased AP-activity and BMP2 levels indicate a loss of function of the osteoblasts. Thus, inhibition of Smad2/3 phosphorylation might positively influence functional activity of osteoblasts in patients with chronically elevated TGF-β₁ levels and thus, could lead to an improved bone healing in vivo.