Using miniaturized radiotelemetry to discover the breeding grounds of the endangered New Zealand Storm Petrel Fregetta maoriana

Identification of breeding sites remains a critical step in species conservation, particularly in procellariiform seabirds whose threat status is of global concern. We designed and conducted an integrative radiotelemetry approach to uncover the breeding grounds of the critically endangered New Zealand Storm Petrel Fregetta maoriana (NZSP), a species considered extinct before its rediscovery in 2003. Solar‐powered automated radio receivers and hand‐held telemetry were used to detect the presence of birds on three island groups in the Hauraki Gulf near Auckland, New Zealand. At least 11 NZSP captured and radiotagged at sea were detected at night near Te Hauturu‐o‐Toi/Little Barrier Island with the detection of an incubating bird leading to the discovery of the first known breeding site for this species. In total, four NZSP breeding burrows were detected under mature forest canopy and three adult NZSP and two NZSP chicks were ringed. Telemetry data indicated NZSP showed strong moonlight avoidance behaviour over the breeding site, had incubation shifts of approximately 5 days and had a breeding season extending from February to June/July, a different season from other Procellariiformes in the region. Radiotelemetry, in combination with rigorously collected field data on species distribution, offers a valuable technique for locating breeding grounds of procellariiform seabirds and gaining insights into breeding biology while minimizing disturbance to sensitive species or damage to fragile habitat. Our study suggests an avenue for other breeding ground searches in one of the most threatened avian Orders, and highlights the general need for information on the location of breeding sites and understanding the breeding biology in data‐deficient birds.     

Identification of P‐glycoprotein co‐fractionating proteins and specific binding partners in rat brain microvessels

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.

     

Methane‐derived carbon flow through microbial communities in arctic lake sediments

Aerobic methane (CH₄) oxidation mitigates CH₄ release and is a significant pathway for carbon and energy flow into aquatic food webs. Arctic lakes are responsible for an increasing proportion of global CH₄ emissions, but CH₄ assimilation into the aquatic food web in arctic lakes is poorly understood. Using stable isotope probing (SIP) based on phospholipid fatty acids (PLFA‐SIP) and DNA (DNA‐SIP), we tracked carbon flow quantitatively from CH₄ into sediment microorganisms from an arctic lake with an active CH₄ seepage. When 0.025 mmol CH₄ g^?1 wet sediment was oxidized, approximately 15.8–32.8% of the CH₄‐derived carbon had been incorporated into microorganisms. This CH₄‐derived carbon equated to up to 5.7% of total primary production estimates for Alaskan arctic lakes. Type I methanotrophs, including Methylomonas, Methylobacter and unclassified Methylococcaceae, were most active at CH₄ oxidation in this arctic lake. With increasing distance from the active CH₄ seepage, a greater diversity of bacteria incorporated CH₄‐derived carbon. Actinomycetes were the most quantitatively important microorganisms involved in secondary feeding on CH₄‐derived carbon. These results showed that CH₄ flows through methanotrophs into the broader microbial community and that type I methanotrophs, methylotrophs and actinomycetes are important organisms involved in using CH₄‐derived carbon in arctic freshwater ecosystems.     

Daily hypoxia increases basal monocyte HSP72 expression in healthy human subjects

Heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. In vitro, research demonstrates HSP72 induction in response to hypoxia. Recently, in vivo, an acute hypoxic exposure (75 min at 2,980 m) was sufficient to induce significant increases in monocyte expressed HSP72 (mHSP72) and a marker of oxidative stress in healthy human subjects. The purpose of the current study was to identify the impact of 10 consecutive days of hypoxic exposures (75 min at 2,980 m) on mHSP72 and erythropoietin (EPO) expression, markers of oxidative stress, and maximal oxygen consumption in graded incremental aerobic exercise. Eight male subjects were exposed to daily normobaric hypoxic exposures for 75 min at 2,980 m for 10 consecutive days, commencing and ceasing at 0930 and 1045, respectively. This stressor was sufficient to induce significant increases in mHSP72, which was significantly elevated from day 2 of the hypoxic exposures until 48 h post-final exposure. Notably, this increase had an initial rapid (30% day on day compared to baseline) and final slow phase (16% day on day compared to baseline) of expression. The authors postulate that 7-day hypoxic exposure in this manner would be sufficient to induce near maximum hypoxia-mediated basal mHSP72 expression. Elevated levels of mHSP72 are associated with acquired thermotolerance and provide cross tolerance to non-related stressors in vivo, the protocol used here may provide a useful tool for elevating mHSP72 in vivo. Aside from these major findings, significant transient daily elevations were seen in a marker of oxidative stress, alongside sustained increases in EPO expression. However, no physiologically significant changes were seen in maximal oxygen consumption or time to exhaustion.     

A missense mutation in the 20S proteasome β2 subunit of Great Danes having harlequin coat patterning

Harlequin is a pigmentary trait of the domestic dog that is controlled by two autosomal loci: the melanosomal gene, SILV, and a modifier gene, harlequin (H), previously localized to chromosome 9. Heterozygosity for a retrotransposon insertion in SILV and a mutation in H causes a pattern of black patches on a white background. Homozygosity for H is embryonic lethal. Fine mapping of the harlequin locus revealed a 25 kb interval wherein all harlequin Great Danes are heterozygous for a common haplotype. This region contains one gene, PSMB7, which encodes the β2 catalytic subunit of the proteasome. Sequence analysis identified a coding variant in exon 2 that segregates with harlequin patterning. The substitution predicts the replacement of a highly conserved valine with a glycine. Described herein is the identification of a naturally-occurring mutation of the ubiquitin proteasome system that is associated with a discernable phenotype of dogs.     

Allocation of maternal- and ejaculate-derived proteins to reproduction in female crickets, Teleogryllus oceanicus

Female fitness has traditionally been thought to be maximized with one or a few matings. More recent research suggests that polyandry, mating with two or more males, can generate an increase in the viability of offspring females produce. However, the mechanism(s) underlying enhanced offspring viability remain largely unknown. The Australian field cricket Teleogryllus oceanicus has proved a useful model for examining the evolutionary significance of polyandry. Embryo viability appears to be associated with a male's investment in accessory gland tissue, implicating a role for seminal fluid. Here, I used amino acids labelled with different radio isotopes to identify proteins manufactured by males and females before they engaged in reproduction. Males incorporated 95% of the radiolabel into the testes, accessory glands and the ejaculate that was transferred to the female at mating. Male ejaculate compounds were incorporated predominantly into the female's somatic tissue. Relatively more female compounds were incorporated into the ovaries and into laid eggs than ejaculate compounds, and relatively fewer female compounds were sequestered in the somatic tissue than ejaculate compounds. The patterns observed suggest that while ejaculate compounds may be incorporated directly into eggs, they are likely to have a larger effect on maternal allocation to offspring.     

Human epidermal growth factor receptor bispecific ligand trap RB200: abrogation of collagen-induced arthritis in combination with tumour necrosis factor blockade

Introduction  

Rheumatoid arthritis (RA) is a chronic disease associated with inflammation and destruction of bone and cartilage. Although inhibition of TNFα is widely used to treat RA, a significant number of patients do not respond to TNFα blockade, and therefore there is a compelling need to continue to identify alternative therapeutic strategies for treating chronic inflammatory diseases such as RA. The anti-epidermal growth factor (anti-EGF) receptor antibody trastuzumab has revolutionised the treatment of patients with EGF receptor-positive breast cancer. Expression of EGF ligands and receptors (known as HER) has also been documented in RA. The highly unique compound RB200 is a bispecific ligand trap that is composed of full-length extracellular domains of HER1 and HER3 EGF receptors. Because of its pan-HER specificity, RB200 inhibits responses mediated by HER1, HER2 and HER3 in vitro and in vivo. The objective of this study was to assess the effect of RB200 combined with TNF blockade in a murine collagen-induced arthritis (CIA) model of RA.     

Eating the strangers within: host control of intracellular bacteria via xenophagy

Many bacterial pathogens rely on an intracellular cycle to ensure their proliferation within infected hosts, through their ability to avoid or circumvent host bactericidal pathways. Recent evidence supports an increasingly important role for the autophagy pathway in innate immune defences against intracellular pathogens, as a mechanism of capture of either cytosol-adapted or vacuolar bacteria that redirect them to the lysosomal compartment for killing. Antibacterial autophagy, also referred to as xenophagy, involves selective recognition of intracellular bacteria and their targeting to the autophagic machinery for degradation. Here we review recent advances in our molecular understanding of these processes, and in how bacteria have adapted to avoid xenophagy or even take advantage of this innate immune process.     

Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.