DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.     

Errors in determining the flow rate of Hirst-type pollen traps

Standardisation of methods of pollen monitoring networks is vital for data quality. In pollen monitoring networks in Europe, the Hirst-type trap is standard. Hirst traps are calibrated with handheld rotameters. We detected a systematic error in the flow rate calibrated by these standard handheld rotameters. We measured the flow rate of 19 Hirst traps from three commercial brands during calibration but also during monitoring. We used three different rotameters supplied by the manufacturers of the traps, respectively. The actual air flow rate was measured using an electronic heat anemometer with negligible air flow resistance. After calibration to 10 l/min, the rotameter was removed, which led to a significant increase in the flow rate in the range of 10.5–17.2 l/min, a systematic error between 5 and 72%. No significant difference was found between the different commercial trap brands. The analysis revealed that the error depended on the type of the rotameter and the individual trap. The error may be explained by the additional air flow resistance of each rotameter. The total resistance of the system—trap plus rotameter—is higher during calibration when the rotameter is held on the inlet compared to the routine monitoring without the rotameter. Depending on the characteristic curve of the suction pump in the trap (fan), the air flow rate increases to values considerably higher than 10 l/min. Thus, monitoring is done under a higher flow rate than that was calibrated. In order to obtain comparable data within a monitoring network, a solution for correction of this systematic error seems advisable, preferably in cooperation with the manufacturers.     

Morphological plasticity in a native freshwater fish from semiarid Australia in response to variable water flows

In fishes, alterations to the natural flow regime are associated with divergence in body shape morphology compared with individuals from unaltered habitats. However, it is unclear whether this morphological divergence is attributable to evolutionary responses to modified flows, or is a result of phenotypic plasticity. Fishes inhabiting arid regions are ideal candidates for studying morphological plasticity as they are frequently exposed to extreme natural hydrological variability. We examined the effect of early exposure to flows on the development of body shape morphology in the western rainbowfish (Melanotaenia australis), a freshwater fish that is native to semiarid northwest Australia. Wild fish were collected from a region (the Hamersley Ranges) where fish in some habitats are subject to altered water flows due to mining activity. The offspring of wild‐caught fish were reared in replicated fast‐flow or slow‐flow channels, and geometric morphometric analyses were used to evaluate variation in fish body shape following 3, 6, 9, and 12 months of exposure. Water flows influenced fish morphology after 6 and 9 months of flow exposure, with fish in fast‐flow environments displaying a more robust body shape than those in slow‐flow habitats. No effect of flow exposure was observed at 3 and 12 months. Fishes also showed significant morphological variation within flow treatments, perhaps due to subtle differences in water flow among the replicate channels. Our findings suggest that early exposure to water flows can induce shifts in body shape morphology in arid zone freshwater fishes. Morphological plasticity may act to buffer arid zone populations from the impacts of anthropogenic activities, but further studies are required to link body shape plasticity with behavioral performance in habitats with modified flows.     

Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.     

Thermal stress induces persistently altered coral reef fish assemblages

Ecological communities are reorganizing in response to warming temperatures. For continuous ocean habitats this reorganization is characterized by large‐scale species redistribution, but for tropical discontinuous habitats such as coral reefs, spatial isolation coupled with strong habitat dependence of fish species imply that turnover and local extinctions are more significant mechanisms. In these systems, transient marine heatwaves are causing coral bleaching and profoundly altering habitat structure, yet despite severe bleaching events becoming more frequent and projections indicating annual severe bleaching by the 2050s at most reefs, long‐term effects on the diversity and structure of fish assemblages remain unclear. Using a 23‐year time series spanning a thermal stress event, we describe and model structural changes and recovery trajectories of fish communities after mass bleaching. Communities changed fundamentally, with the new emergent communities dominated by herbivores and persisting for >15 years, a period exceeding realized and projected intervals between thermal stress events on coral reefs. Reefs which shifted to macroalgal states had the lowest species richness and highest compositional dissimilarity, whereas reefs where live coral recovered exceeded prebleaching fish richness, but remained dissimilar to prebleaching compositions. Given realized and projected frequencies of bleaching events, our results show that fish communities historically associated with coral reefs will not re‐establish, requiring substantial adaptation by managers and resource users.     

Facultative adjustment of pre-fledging mass loss by nestling swifts preparing for flight

Nestling birds often maintain nutritional reserves to ensure continual growth during interruptions in parental provisioning. However, mass-dependent flight costs require the loss of excess mass before fledging. Here we test whether individual variable mass loss prior to fledging is controlled through facultative adjustments by nestlings, or whether it reflects physiologically inflexible developmental schedules. We show that in the face of natural and experimental variation in nestling body mass and wing length, swifts always achieve very similar wing loadings (body mass per wing area) prior to fledging, presumably because this represents the optimum for flight. Experimental weights (approx. 5% body mass) temporarily attached to nestlings caused additional reductions in mass, such that final wing loadings still matched those of control siblings. Experimental reductions in nestling wing length (approx. 5% trimmed from feather tips) resulted in similar additional mass reductions, allowing wing loadings at fledging to approach control levels. We suggest that nestlings may assess their body mass relative to wing area via wing flapping and special 'push-ups' (on the tips of extended wings) performed in the nest. Thus, by facultatively adjusting body mass, but not wing growth, nestling swifts are always able to fledge with aerodynamically appropriate wing loadings.     

PI 3-kinase p110beta: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.     

Unprecedented Mass Bleaching and Loss of Coral across 12° of Latitude in Western Australia in 2010–11

Background

Globally, coral bleaching has been responsible for a significant decline in both coral cover and diversity over the past two decades. During the summer of 2010–11, anomalous large-scale ocean warming induced unprecedented levels of coral bleaching accompanied by substantial storminess across more than 12° of latitude and 1200 kilometers of coastline in Western Australia (WA).

Methodology/Principal Findings

Extreme La-Niña conditions caused extensive warming of waters and drove considerable storminess and cyclonic activity across WA from October 2010 to May 2011. Satellite-derived sea surface temperature measurements recorded anomalies of up to 5°C above long-term averages. Benthic surveys quantified the extent of bleaching at 10 locations across four regions from tropical to temperate waters. Bleaching was recorded in all locations across regions and ranged between 17% (±5.5) in the temperate Perth region, to 95% (±3.5) in the Exmouth Gulf of the tropical Ningaloo region. Coincident with high levels of bleaching, three cyclones passed in close proximity to study locations around the time of peak temperatures. Follow-up surveys revealed spatial heterogeneity in coral cover change with four of ten locations recording significant loss of coral cover. Relative decreases ranged between 22%–83.9% of total coral cover, with the greatest losses in the Exmouth Gulf.

Conclusions/Significance

The anomalous thermal stress of 2010–11 induced mass bleaching of corals along central and southern WA coral reefs. Significant coral bleaching was observed at multiple locations across the tropical-temperate divide spanning more than 1200 km of coastline. Resultant spatially patchy loss of coral cover under widespread and high levels of bleaching and cyclonic activity, suggests a degree of resilience for WA coral communities. However, the spatial extent of bleaching casts some doubt over hypotheses suggesting that future impacts to coral reefs under forecast warming regimes may in part be mitigated by southern thermal refugia.     

A study on the postrelease behaviour and habitat preferences of black rhinos (Diceros bicornis) reintroduced into a fenced reserve in Namibia

Translocations and reintroductions are key elements for the population management of the critically endangered black rhino (Diceros bicornis, Linnaeus, 1758). In this study, we investigated the postrelease behaviour and habitat preferences of a black rhino starter group (n = 4) on the individual level. The animals were reintroduced to a fenced game reserve (87 km²) in North‐Central Namibia 1 year prior to our study. We used camera traps and very high frequency (VHF) radiotelemetry to examine the animals' temporal and spatial behaviour over a period of 4 months at transition between wet and dry seasons. Our results underline a peak in drinking activity and waterhole visits occurring between 7 p.m. and 8 p.m. We found a shift in intensity in drinking activity during the period of the study. Satellite‐based woody cover estimations only suggest positive correlations between the density of woody cover and favoured black rhino habitat types. Although the area seems suitable to facilitate breeding success of this starter group, it does not support a self‐sustaining population. However, black rhinos were already successfully reintroduced to several additional fenced reserves in this region. The selective opening of fences in the future could help to enable genetic exchange between currently isolated groups of rhinos.     

Diarrhea, pneumonia, and infectious disease mortality in children aged 5 to 14 years in India

Background

Little is known about the causes of death in children in India after age five years. The objective of this study is to provide the first ever direct national and sub-national estimates of infectious disease mortality in Indian children aged 5 to 14 years.

Methods

A verbal autopsy based assessment of 3 855 deaths is children aged 5 to 14 years from a nationally representative survey of deaths occurring in 2001–03 in 1·1 million homes in India.

Results

Infectious diseases accounted for 58% of all deaths among children aged 5 to 14 years. About 18% of deaths were due to diarrheal diseases, 10% due to pneumonia, 8% due to central nervous system infections, 4% due to measles, and 12% due to other infectious diseases. Nationally, in 2005 about 59 000 and 34 000 children aged 5 to 14 years died from diarrheal diseases and pneumonia, corresponding to mortality of 24·1 and 13·9 per 100 000 respectively. Mortality was nearly 50% higher in girls than in boys for both diarrheal diseases and pneumonia.

Conclusions

Approximately 60% of all deaths in this age group are due to infectious diseases and nearly half of these deaths are due to diarrheal diseases and pneumonia. Mortality in this age group from infectious diseases, and diarrhea in particular, is much higher than previously estimated.