Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal ⁵⁹Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.     

Cancer incidence and mortality in people aged less than 75 years: Changes in Australia over the period 1987–2007

BackgroundAustralia has one of the highest rates of cancer incidence worldwide and, despite improving survival, cancer continues to be a major public health problem. Our aim was to provide simple summary measures of changes in cancer mortality and incidence in Australia so that progress and areas for improvement in cancer control can be identified.MethodsWe used national data on cancer deaths and newly registered cancer cases and compared expected and observed numbers of deaths and cases diagnosed in 2007. The expected numbers were obtained by applying 1987 age–sex specific rates (average of 1986–1988) directly to the 2007 population. The observed numbers of deaths and incident cases were calculated for 2007 (average of 2006–2008). We limited the analyses to people aged less than 75 years.ResultsThere was a 28% fall in cancer mortality (7827 fewer deaths in 2007 vs. 1987) and a 21% increase in new cancer diagnoses (13,012 more diagnosed cases in 2007). The greatest reductions in deaths were for cancers of the lung in males (?2259), bowel (?1797), breast (?773) and stomach (?577). Other notable falls were for cancers of the prostate (?295), cervix (?242) and non-Hodgkin lymphoma (?240). Only small or no changes occurred in mortality for cancers of the lung (female only), pancreas, brain and related, oesophagus and thyroid, with an increase in liver cancer (267). Cancer types that showed the greatest increase in incident cases were cancers of the prostate (10,245), breast (2736), other cancers (1353), melanoma (1138) and thyroid (1107), while falls were seen for cancers of the lung (?1705), bladder (?1110) and unknown primary (?904).ConclusionsThe reduction in mortality indicates that prevention strategies, improvements in cancer treatment, and screening programmes have made significant contributions to cancer control in Australia since 1987. The rise in incidence is partly due to diagnoses being brought forward by technological improvements and increased coverage of screening and early diagnostic testing.     

Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.     

Phthalates Are Metabolised by Primary Thyroid Cell Cultures but Have Limited Influence on Selected Thyroid Cell Functions In Vitro

Phthalates are plasticisers added to a wide variety of products, resulting in measurable exposure of humans. They are suspected to disrupt the thyroid axis as epidemiological studies suggest an influence on the peripheral thyroid hormone concentration. The mechanism is still unknown as only few in vitro studies within this area exist. The aim of the present study was to investigate the influence of three phthalate diesters (di-ethyl phthalate, di-n-butyl phthalate (DnBP), di-(2-ethylhexyl) phthalate (DEHP)) and two monoesters (mono-n-butyl phthalate and mono-(2-ethylhexyl) phthalate (MEHP)) on the differentiated function of primary human thyroid cell cultures. Also, the kinetics of phthalate metabolism were investigated. DEHP and its monoester, MEHP, both had an inhibitory influence on 3''-5''-cyclic adenosine monophosphate secretion from the cells, and MEHP also on thyroglobulin (Tg) secretion from the cells. Results of the lactate dehydrogenase-measurements indicated that the MEHP-mediated influence was caused by cell death. No influence on gene expression of thyroid specific genes (Tg, thyroid peroxidase, sodium iodine symporter and thyroid stimulating hormone receptor) by any of the investigated diesters could be demonstrated. All phthalate diesters were metabolised to the respective monoester, however with a fall in efficiency for high concentrations of the larger diesters DnBP and DEHP. In conclusion, human thyroid cells were able to metabolise phthalates but this phthalate-exposure did not appear to substantially influence selected functions of these cells.     

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the bla_CMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare bla_CMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying bla_CMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying bla_CMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of bla_CMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing bla_CMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.     

Objectively Measured Physical Activity in European Adults: Cross-Sectional Findings from the Food4Me Study

Background

Comparisons of objectively measured physical activity (PA) between residents of European countries measured concurrently with the same protocol are lacking. We aimed to compare PA between the seven European countries involved in the Food4Me Study, using accelerometer data collected remotely via the Internet.

Methods

Of the 1607 participants recruited, 1287 (539 men and 748 women) provided at least 3 weekdays and 2 weekend days of valid accelerometer data (TracmorD) at baseline and were included in the present analyses.

Results

Men were significantly more active than women (physical activity level = 1.74 vs. 1.70, p < 0.001). Time spent in light PA and moderate PA differed significantly between countries but only for women. Adherence to the World Health Organization recommendation to accumulate at least 150 min of moderate-equivalent PA weekly was similar between countries for men (range: 54–65%) but differed significantly between countries for women (range: 26–49%). Prevalence estimates decreased substantially for men and women in all seven countries when PA guidelines were defined as achieving 30 min of moderate and vigorous PA per day.

Conclusions

We were able to obtain valid accelerometer data in real time via the Internet from 80% of participants. Although our estimates are higher compared with data from Sweden, Norway, Portugal and the US, there is room for improvement in PA for all countries involved in the Food4Me Study.     

Hydrocarbons Are Essential for Optimal Cell Size,Division, and Growth of Cyanobacteria

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.Cyanobacteria (oxygenic photosynthetic bacteria) are found in nearly every environment on Earth and are major contributors to global carbon and nitrogen fixation (Galloway et al., 2004; Zwirglmaier et al., 2008). They are distinguished among prokaryotes in containing multiple internal thylakoid membranes, the site of photosynthesis, and a large protein compartment, the carboxysome, involved in carbon fixation. Despite these extra features, cyanobacteria can be as small as 0.6 µm in diameter (Raven, 1998).All cyanobacteria with sequenced genomes encode the pathway for the biosynthesis of hydrocarbons, implying an important, although as-yet-undefined, role for these compounds (Lea-Smith et al., 2015). The major forms are C15-C19 alkanes and alkenes, which can be synthesized from fatty acyl-acyl-carrier proteins (ACPs) by one or other of two separate pathways (Fig. 1; Schirmer et al., 2010; Mendez-Perez et al., 2011). The majority of species produce alkanes and alkenes via acyl-ACP reductase (FAR) and aldehyde deformylating oxygenase (FAD; Schirmer et al., 2010; Li et al., 2012; Coates et al., 2014; Lea-Smith et al., 2015). Cyanobacterial species lacking the FAR/FAD pathway synthesize alkenes via olefin synthase (Ols; Mendez-Perez et al., 2011; Coates et al., 2014; Lea-Smith et al., 2015). This suggests that hydrocarbons produced by either pathway serve a similar role in the cell. Homologs of FAR/FAD or Ols are not present in other bacteria or plant and algal species. However, C15-C17 alkanes and alkenes, synthesized by an alternate, uncharacterized pathway, were recently detected in a range of green microalgae, including Chlamydomonas reinhardtii, Chlorella variabilis NC64A, and several Nannochloropsis species (Sorigué et al., 2016). In C. reinhardtii, hydrocarbons were primarily localized to the chloroplast, which originated in evolution from a cyanobacterium that was engulfed by a host organism (Howe et al., 2008). Hydrocarbons may therefore have a similar role in cyanobacteria, some green microalgae species, and possibly a broader range of photosynthetic organisms.Open in a separate windowFigure 1.Hydrocarbon biosynthesis is encoded in all sequenced cyanobacteria. Detailed are the two hydrocarbon biosynthetic pathways, indicated in blue and red, respectively, in cyanobacteria. The number of species encoding the enzymes in each pathway is indicated.Hydrocarbons act as antidesiccants, waterproofing agents, and signaling molecules in insects (Howard and Blomquist, 2005) and prevent water loss, ensure pollen viability, and influence pathogen interactions in plants (Kosma et al., 2009; Bourdenx et al., 2011). However, the function of hydrocarbons in cyanobacteria has not been determined. Characterization of cyanobacterial hydrocarbon biosynthesis pathways has provided the basis for investigating synthetic microbial biofuel systems, which may be a renewable substitute for fossil fuels (Schirmer et al., 2010; Choi and Lee, 2013; Howard et al., 2013). However, secretion of long-chain hydrocarbons from the cell into the medium, which is likely essential for commercially viable production, has not been observed in the absence of a membrane solubilization agent (Schirmer et al., 2010; Tan et al., 2011). Cyanobacterial hydrocarbons also have a significant environmental role. Due to the abundance of cyanobacteria in the environment, hydrocarbon production is considerable, with hundreds of millions of tons released into the ocean per annum following cell death (Lea-Smith et al., 2015). This production may be sufficient to sustain populations of hydrocarbon-degrading bacteria, which can then play an important role in consuming anthropogenic oil spills (Lea-Smith et al., 2015).Here, we investigated the cellular location and role of hydrocarbons in both spherical Synechocystis sp. PCC 6803 (Synechocystis) and rod-shaped Synechococcus sp. PCC 7002 (Synechococcus) cells. We developed a model of the cyanobacterial membrane, which indicated that hydrocarbons aggregate in the middle of the lipid bilayer and, when present at levels observed in cells, lead to membrane swelling associated with pools of hydrocarbon. This suggested that alkanes may facilitate membrane curvature. In vivo measurements of Synechococcus thylakoid membrane conformation are consistent with this model.     

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.Seeds play a major role in keeping continuity between successive generations (Esau, 1977) and are key for the distribution and evolution (Moles et al., 2005) of higher plants. Fertile seeds carry an embryo and may contain nutrient storage tissues in cotyledons, endosperm, and/or perisperm, supporting germination and seedling development at early developmental stages. Although this is true for all seed plants, various traits of seeds, such as size, shape, weight, and chemical composition, can be very different between plant species or accessions. For example, the Arabidopsis (Arabidopsis thaliana) accession Cape Verde Islands was reported to yield on average 40% fewer seeds than Landsberg erecta, but they are almost twice as heavy (Alonso-Blanco et al., 1999). Considering today’s plant species, single-seed mass may vary over a range of 11.5 orders of magnitude (Moles et al., 2005). Seed mass is under strong genetic control, whereas the total number of seeds of a plant is largely affected by the environment (Paul-Victor and Turnbull, 2009). It has been demonstrated that the size, mass, and shape of Arabidopsis seeds may be regulated by brassinosteroid (Jiang et al., 2013), and it was shown recently that seed size in rice (Oryza sativa) can be influenced by the epiallele Epi-rav6 (Zhang et al., 2015). The ability of plants to switch between small and larger seeds may be understood as an adaptation to novel environments (Igea et al., 2016). However, it is still not fully understood whether, or to what extent, the variability of seed traits within plant species or genotypes has an impact on the development and further performance of a plant.When comparing biometric seed data of different dimensions such as length (one-dimensional), projected area (two-dimensional [2D]), or volume and mass (both three-dimensional [3D]), one can argue that mass is the most relevant parameter as a proxy for the amount of reserves a seed provides for the offspring. This might be true even when considering that the type of reserves, such as proteins, carbohydrates, or lipids (Rolletschek et al., 2015), and also different seed tissues, such as seed coat, embryo, or endosperm, may contribute differently to seed mass (Alonso-Blanco et al., 1999). While seed mass and time to germination (radicle protrusion) do not necessarily correlate (Norden et al., 2009), in particular under greenhouse conditions, higher seed mass may be advantageous for seedling establishment under adverse environmental conditions (Moles et al., 2005). For example, shade-tolerant species showed largely higher seed masses than cogeneric species growing in open habitats, indicating that seedlings under low-light conditions need more reserves than under good light (Salisbury, 1974). Seedlings of wild radish (Raphanus raphanistrum) emerged more likely from heavier seeds than from small seeds under field conditions but not in the greenhouse (Stanton, 1984), and for Arabidopsis, seed mass was reported to be higher in populations growing naturally at higher altitudes taken as a proxy for harsher conditions (Montesinos-Navarro et al., 2011).Seed mass can be measured individually (Stanton, 1984), but it is generally collected as an average value of batches of 50 to 1,000 seeds (Jako et al., 2001; Jofuku et al., 2005; Montesinos-Navarro et al., 2011; Tanabata et al., 2012). Alternatively, 2D scans are analyzed to determine parameters such as seed length, width, area, and perimeter length as a measure for seed size (Tanabata et al., 2012). This approach can be implemented in high-throughput facilities to obtain projected areas of seed grains combined with genome-wide association studies (Yang et al., 2014). Although projected seed area can easily be measured with a common office scanner (Herridge et al., 2011; Tanabata et al., 2012; Moore et al., 2013), it is not necessarily a precise or reliable measure of the true seed size because it may depend on the shape (Alonso-Blanco et al., 1999) and the orientation of a seed at scan (see “Results”). These issues also apply when using 2D projections to calculate length-to-width ratios as a simple shape factor (Tanabata et al., 2012). Projected seed area also has been used to calculate seed mass, assuming a fixed relationship between these parameters (de Jong et al., 2011; Herridge et al., 2011). This may hold with sufficient accuracy when averaging a large number of seeds but might be misleading when considering individual seeds.From a physical point of view, volume should be a much better proxy for mass than 2D traits. Although it has been stated that for 65 species analyzed seed masses can be compared easily with seed volumes (Moles et al., 2005), it is not clear how these seed volumes were determined. Volumes can be assessed using advanced methods such as x-ray computed tomography (CT) on fruits (Stuppy et al., 2003) or synchrotron radiation x-ray tomographic microscopy applied in paleobiological studies (e.g. on fruits and seed; Friis et al., 2014). Nuclear magnetic resonance (NMR) methods are used to measure water uptake in kidney beans (Phaseolus vulgaris) and adzuki beans (Vigna angularis; Kikuchi et al., 2006) or to estimate seed weight and content (Borisjuk et al., 2011; Rolletschek et al., 2015) rather than volumes. To our best knowledge, affordable methods to measure seed volumes directly are not achievable so far. For that reason, we have set up a volume-carving method for 3D seed shape reconstruction that is described briefly here and in more detail in a recent publication (Roussel et al., 2016).While traits derived from scanning procedures can easily be assigned to individual seeds (Herridge et al., 2011), further handling and processing of phenotyped single seeds is not as simple, in particular for tiny ones like those of Arabidopsis. The aim of this work was to develop an automated seed-handling system that can analyze single seeds of very different sizes or shapes, from Arabidopsis seeds up to barley (Hordeum vulgare) seeds or even bigger. The phenoSeeder system is designed to pick and place seeds, to achieve basic morphometric traits (one-dimensional and 2D data from projections, 3D reconstruction data, and mass) of each individual seed, and to store all analyzed seed traits in a database. Another goal is to use phenoSeeder for seed-to-plant tracking approaches and to analyze whether, or which, particular seed traits have an impact on plant development and performance under various environmental conditions. We describe the main features of the phenoSeeder technology and present results obtained with seeds of three accessions of Arabidopsis, rapeseed (Brassica napus), and barley, respectively. When analyzing the data, we focused particularly on correlations between projected seed area, seed volume, and seed mass, with the hypothesis that the respective seed volume may better correlate with mass than the projected area.