Metabolomic Analysis of the Effect of Postnatal Hypoxia on the Retina in a Newly Born Piglet Model

The availability of reliable biomarkers of brain injury secondary to birth asphyxia could substantially improve clinical grading, therapeutic intervention strategies, and prognosis. In this study, changes in the metabolome of retinal tissue caused by profound hypoxia in an established neonatal piglet model were investigated using an ultra performance liquid chromatography – quadrupole time of flight mass spectrometry (UPLC-QTOFMS) untargeted metabolomic approach, which included Partial Least Squares – Discriminant Analysis (PLSDA) multivariate data analysis. The initial identification of a set of discriminant metabolites from UPLC-QTOFMS data was confirmed by target UPLC-MS/MS and allowed the selection of endogenous CDP-choline as a promising candidate biomarker for hypoxia-derived brain damage assessing intensity of retinal hypoxia. Results from this study will foster further research on CDP-choline changes occurring during resuscitation.     

Optimal orientation in branched cytoskeletal networks

Actin cytoskeletal protrusions in crawling cells, or lamellipodia, exhibit various morphological properties such as two characteristic peaks in the distribution of filament orientation with respect to the leading edge. To understand these properties, using the dendritic nucleation model as a basis for cytoskeletal restructuring, a kinetic-population model with orientational-dependent branching (birth) and capping (death) is constructed and analyzed. Optimizing for growth yields a relation between the branch angle and filament orientation that explains the two characteristic peaks. The model also exhibits a subdominant population that allows for more accurate modeling of recent measurements of filamentous actin density along the leading edge of lamellipodia in keratocytes. Finally, we explore the relationship between orientational and spatial organization of filamentous actin in lamellipodia and address recent observations of a prevalence of overlapping filaments to branched filaments-a finding that is claimed to be in contradiction with the dendritic nucleation model.     

Temporal and Spatial Patterns in Inputs and Stock of Organic Matter in Savannah Streams of Central Brazil

Coarse particulate organic matter (CPOM) inputs from riparian vegetation into streams and CPOM benthic stock vary naturally in space and time, but most studies in the tropical savannah (Cerrado) have been done over a small temporal scale (<1 year), which does not allow for the determination of inter-annual patterns. We found that CPOM collected over two years differed temporally and spatially, whereas there was no significant variation between years for the benthic stock, which indicates high stability in the energy balance of streams. The largest monthly inputs occurred between August and October, at the end of the dry season and the onset of the rainy season, which was partially explained by precipitation. Other factors such as photoperiod, which was not studied, could also have important roles in this pattern. Spatial differences in CPOM between streams were attributed to topography and channel morphology. The plant density was lowest in the stream with a more irregular topography and a deeper channel, which results in drier riparian soil. The benthic stock was highest in the stream with a flat channel, where the lower water speed facilitates the accumulation of CPOM in the stream bed. Inter-annual differences in CPOM were attributed more to the differences in the beginning of the dry and wet periods between years than to the average values of precipitation. Longer-term studies are needed to clarify this temporal pattern.     

Duplicate marker loci can result in incorrect locus orders on linkage maps

Genetic linkage maps, constructed from multi-locus recombination data, are the basis for many applications of molecular markers. For the successful employment of a linkage map, it is essential that the linear order of loci on a chromosome is correct. The objectives of this theoretical study were to (1) investigate the occurrence of incorrect locus orders caused by duplicate marker loci, (2) develop a statistical test for the detection of duplicate markers, and (3) discuss the implications for practical applications of linkage maps. We derived conditions, under which incorrect locus orders do or do not occur with duplicate marker loci for the general case of n markers on a chromosome in a BC₁ mapping population. We further illustrated these conditions numerically for the special case of four markers. On the basis of the extent of segregation distortion, an exact test for the presence of duplicate marker loci was suggested and its power was investigated numerically. Incorrect locus orders caused by duplicate marker loci can (1) negatively affect the assignment of target genes to chromosome regions in a map-based cloning experiment, (2) hinder indirect selection for a favorable allele at a quantitative trait locus, and (3) decrease the efficiency of reducing the length of the chromosome segment attached to a target gene in marker-assisted backcrossing.Communicated by G. WenzelM. Frisch and M. Quint contributed equally to this article.     

Failure of CBA/N mice to respond to thymus-dependent and thymus-independent phosphorylcholine antigens.

CBA/N mice have an X-linked defect of B lymphocyte differentiation. We have studied their responsiveness to different antigenic forms of the phosphorylcholine (PC) epitope which induce IgM responses of restricted heterogeneity. Although previous work had emphasized a defect of T.I. responses in CBA/N we found that both T.D. and T.I. anti-PC responses are severely depressed in CBA/N, in spite of the use of bacterial adjuvants or repeated immunizations; however, with certain immunization protocols we could detect in CBA/N direct and indirect PFC whose specificity was not limited to PC but included the PC-protein bridge. Transfer experiments involving normal or irradiated immunodeficient F₁ hybrid males as recipients of normal spleen or bone marrow cells failed to implicate suppressive mechanisms in the unresponsiveness to PC antigens. Our results indicate that the defect in antigen-induced humoral responses in CBA/N mice is not simply related to the thymus-dependence of the antigen used but rather a consequence of selective maturational defects of subpopulations of B lymphocytes and of subsets within each subpopulation.     

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.     

The Infection of Soybean Leaves by Phakopsora pachyrhizi during Conditions of Discontinuous Wetness

The ability of Phakopsora pachyrhizi to cause infection under conditions of discontinuous wetness was investigated. In in vitro experiments, droplets of a uredospore suspension were deposited onto the surface of polystyrene. After an initial wetting period of either 1, 2 or 4 h, the drops were dried for different time intervals and then the wetness was restored for 11, 10 or 8 h. Germination and appressorium formation were evaluated. In in vivo experiments, soybean plants were inoculated with a uredospore suspension. Leaf wetness was interrupted for 1, 3 or 6 h after initial wetting periods of 1, 2 or 4 h. Then, the wetting was re‐established for 11, 10 or 8 h, respectively. Rust severity was evaluated 14 days after inoculation. The germination of the spores and the formation of the appressoria on the soybean leaves after different periods of wetness were also quantified in vivo by scanning electron microscopy. P. pachyrhizi showed a high infective capacity during short periods of time. An interruption of wetness after 1 h caused average reductions in germination from 56 to 75% and in appressorium formation from 84 to 96%. Rust severity was lower in all of the in vivo treatments with discontinuous wetness when compared to the control plants. Rust severity was zero when the interruption of wetness occurred 4 h after the initial wetting. Wetting interruptions after 1 and 2 h reduced the average rust severity by 83 and 77%, respectively. The germination of the uredospores on the soybean leaves occurred after 2 h of wetness, with a maximum germination appearing after 4 h of wetness. Wetness interruption affected mainly the spores that had initiated the germination.