Assessment of Oxidative Damage to Proteins and DNA in Urine of Newborn Infants by a Validated UPLC-MS/MS Approach

The assessment of oxidative stress is highly relevant in clinical Perinatology as it is associated to adverse outcomes in newborn infants. This study summarizes results from the validation of an Ultra Performance Liquid Chromatography–tandem Mass Spectrometry (UPLC-MS/MS) method for the simultaneous quantification of the urinary concentrations of a set of endogenous biomarkers, capable to provide a valid snapshot of the oxidative stress status applicable in human clinical trials, especially in the field of Perinatology. The set of analytes included are phenylalanine (Phe), para-tyrosine (p-Tyr), ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-NO₂-tyrosine (3NO₂-Tyr), 3-Cl-tyrosine (3Cl-Tyr), 2′-deoxyguanosine (2dG) and 8-hydroxy-2′-deoxyguanosine (8OHdG). Following the FDA-based guidelines, appropriate levels of accuracy and precision, as well as adequate levels of sensitivity with limits of detection (LODs) in the low nanomolar (nmol/L) range were confirmed after method validation. The validity of the proposed UPLC-MS/MS method was assessed by analysing urine samples from a clinical trial in extremely low birth weight (ELBW) infants randomized to be resuscitated with two different initial inspiratory fractions of oxygen.     

Mysterious bio-duck sound attributed to the Antarctic minke whale (Balaenoptera bonaerensis)

For decades, the bio-duck sound has been recorded in the Southern Ocean, but the animal producing it has remained a mystery. Heard mainly during austral winter in the Southern Ocean, this ubiquitous sound has been recorded in Antarctic waters and contemporaneously off the Australian west coast. Here, we present conclusive evidence that the bio-duck sound is produced by Antarctic minke whales (Balaenoptera bonaerensis). We analysed data from multi-sensor acoustic recording tags that included intense bio-duck sounds as well as singular downsweeps that have previously been attributed to this species. This finding allows the interpretation of a wealth of long-term acoustic recordings for this previously acoustically concealed species, which will improve our understanding of the distribution, abundance and behaviour of Antarctic minke whales. This is critical information for a species that inhabits a difficult to access sea-ice environment that is changing rapidly in some regions and has been the subject of contentious lethal sampling efforts and ongoing international legal action.     

Wet and dry season diets of the Endangered Lemur catta (ring‐tailed lemur) in two mountainous rocky outcrop forest fragments in south‐central Madagascar

Lemur catta's ability to consume a wide variety of plant foods is a key to this species' survival in a time of ecological crisis across its geographic range. We examined seasonal diet variability of L. catta groups inhabiting two rocky outcrop fragments in south‐central Madagascar: Anja Reserve and Tsaranoro Valley forest. Leaves and fruit of Melia azedarach were a keystone resource for Anja lemurs in wet and dry seasons. At Tsaranoro, L. catta relied on M. azedarach and Ficus spp. in dry season, but during the wet season, neither was a dominant food resource. Top food species at both sites differed markedly from those consumed by L. catta in other habitats. At Tsaranoro, a greater proportion of lemurs engaged in feeding during the dry season compared with wet season. We attribute this to resource scarcity during dry season, when greater feeding effort is needed to maintain energy requirements. Because M. azedarach is ubiquitous throughout Anja Reserve, producing fruit and leaves year‐round, Anja lemurs can meet energy requirements with little seasonal adjustment in feeding activity. L. catta's IUCN status has been upgraded to Endangered, thus, greater insight into its diet flexibility and ability to survive on introduced plant species, can inform conservation plans in remaining wild habitats and ex situ programs.     

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

Plasmodium falciparum virulence is linked to its ability to sequester in post‐capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9‐96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence‐associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co‐exported with PfEMP1 into the host cell via vesicle‐like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

Denitrification Potential in Lake Sediment Increases Across a Gradient of Catchment Agriculture

Intensification of catchment agriculture has increased nutrient loads and accelerated eutrophication in some lakes, often resulting in episodic harmful algal blooms or prolonged periods of anoxia. The influence of catchment agriculture on lake sediment denitrification capacity as a nitrogen (N) removal mechanism in lakes is largely unknown, particularly in contrast to research on denitrification in agricultural streams and rivers. We measured denitrification enzyme activity (DEA) to assess sediment denitrification potential in seven monomictic and three polymictic lakes that range in the proportion of agriculture in the catchment from 3 to 96% to determine if there is a link between agricultural land use in the lake catchment and sediment denitrification potential. We collected sediment cores for DEA measurements over 3 weeks in austral spring 2008 (October–November). Lake Okaro, with 96% catchment agriculture, had approximately 15 times higher DEA than Lake Tikitapu, with 3% catchment agriculture (232.2 ± 55.9 vs. 15.9 ± 4.5 μg N gAFDM⁻¹ h⁻¹, respectively). Additionally, sediment denitrification potential increased with the proportion of catchment in agriculture (R ² = 0.85, P < 0.001). Our data suggest that lakes retain a high capacity to remove excess N via denitrification under increasing N loads from higher proportions of catchment agriculture. However, evidence from the literature suggests that despite a high capacity for denitrification and longer water residence times, lakes with high N loads will still remove a smaller proportion of their N load. Lakes have a denitrification potential that reflects the condition of the lake catchment, but more measurements of in situ denitrification rates across lake catchments is necessary to determine if this capacity translates to high N removal rates.     

Structural Relaxation During Drying and Rehydration of Food Materials��the Water Effect and the Origin of Hysteresis

The state of water in foodstuffs is a guiding principle in food design, and the equilibrium concept of water activity (Aw) is ubiquitous. It is regarded as a primary variable or “hurdle” in preservation technology, and a key variable influencing chemical reaction during storage. However, the amount of water in any system differs as function of water activity depending whether it is determined by water sorption or desorption. Even though this hysteresis behaviour has already been described in the literature, no physical interpretation of its origin has yet been proposed with respect to detailed molecular organisation. This work shows, for two different food powders, gluten and a milk-based product that the hysteresis disappears when either go through their glass transition. A more complete DSC analysis for gluten during different sorption/desorption cycles demonstrates that the hysteresis is dependent on the ageing of the material, which evolves in the glassy state and is induced by structural relaxation.     

Septins at the annulus of mammalian sperm

The annulus is an electron-dense ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Proteins from the septin family have been shown to localize to the annulus. A septin complex is assembled early in spermiogenesis with the cochaperone DNAJB13 and, in mature sperm, associates with Testis Anion Transporter 1; SLC26A8 (Tat1), a transmembrane protein of the SLC26 family. Studies in mice have shown that the annulus acts as a barrier to protein diffusion and controls correct organization of the midpiece. Consistent with these findings, absence of the annulus is associated with flagellum differentiation defects and asthenozoospermia in humans.     

IL-13Rα2-Targeted Therapy Escapees: Biologic and Therapeutic Implications

Glioblastoma multiforme (GBM) overexpresses interleukin 13 receptor α2 (IL-13Rα2), a tumor-restricted receptor that is not present in normal brain. We and others have created targeted therapies that specifically eradicate tumors expressing this promising tumor-restricted biomarker. As these therapies head toward clinical implementation, it is critical to explore mechanisms of potential resistance. We therefore used a potent IL-13Rα2-targeted bacterial cytotoxin to select for naturally occurring "escapee" cells from three different IL-13Rα2-expressing GBM cell lines. We found that these side populations of escapee cells had significantly decreased IL-13Rα2 expression. We examined clinically relevant biologic characteristics of escapee cell lines compared to their parental cell lines and found that they had similar proliferation rates and equal sensitivity to temozolomide and radiation, the standard therapies given to GBM patients. In contrast, our escapee cell lines were less likely to form colonies in culture and migrated more slowly in wound healing assays. Furthermore, we found that escapee cells formed significantly less neurospheres in vitro, suggesting that IL-13Rα2-targeted therapy preferentially targeted the "stem-like" cell population and possibly indicating decreased tumorigenicity in vivo. We therefore tested escapee cells for in vivo tumorigenicity and found that they were significantly less tumorigenic in both subcutaneous and intracranial mouse models compared to matching parental cells. These data, for the first time, establish and characterize the clinically relevant biologic properties of IL-13Rα2-targeted therapy escapees and suggest that these cells may have less malignant characteristics than parental tumors.     

A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.