Intrapatient functional clonality deconvoluted by coupling intracellular flow cytometry and next-generation sequencing in human leukemia.

The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte-macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.

Clinical review of Hendra virus infection in 11 horses in New South Wales, Australia.

CASE SERIES: Between 2006 and 2012, there were 11 horses diagnosed with Hendra virus (HeV) on 9 independent premises in New South Wales (NSW). We defined a case of HeV as premises where one or more horses were confirmed to be infected with HeV by PCR. All the cases occurred in the north-eastern region of NSW. In 8 of the 9 cases, infection occurred within 2 months over the winter of 2011. With no exception, the affected horses were kept at pasture on properties visited by flying foxes. Of the 11 horses testing positive for HeV, 5 had an association with a fence, with the horses dead or dying on a fence line. In the majority of cases, disease was an acute illness leading to death within 48 h. When signs of disease were observed, neurological signs predominated. There was limited spread to in-contact horses, with only two properties having more than one horse affected. There was significant variation in the sampling strategies undertaken by veterinarians. CONCLUSION: Caution is needed to interpret a negative diagnosis when only swabs have been collected.

Light-dependent maintenance of hydraulic function in mangrove branches: do xylary chloroplasts play a role in embolism repair?

• To clarify the role of branch photosynthesis in tree functioning, the presence and function of chloroplasts in branch xylem tissue were studied in a diverse range of mangrove species growing in Australia. • The presence of xylary chloroplasts was observed via chlorophyll fluorescence of transverse sections. Paired, attached branches were selected to study the effects of covering branches with aluminium foil on the gas exchange characteristics of leaves and the hydraulic conductivity of branches. • Xylary chloroplasts occurred in all species, but were differently distributed among living cell types in the xylem. Covering stems altered the gas exchange characteristics of leaves, such that water-use efficiency was greater in exposed leaves of covered than of uncovered branches. • Leaf-specific hydraulic conductivity of stems was lower in covered than in uncovered branches, implicating stem photosynthesis in the maintenance of hydraulic function. Given their proximity to xylem vessels, we suggest that xylary chloroplasts may play a role in light-dependent repair of embolized xylem vessels.

Do thick leaves avoid thermal damage in critically low wind speeds?

Transient lulls in air movement are rarely measured, but can cause leaf temperature to rise rapidly to critical levels. The high heat capacity of thick leaves can damp this rapid change in temperature. However, little is known about the extent to which increased leaf thickness can reduce thermal damage, or how thick leaves would need to be to have biological significance. We evaluated quantitatively the contribution of small increases in leaf thickness to the reduction in thermal damage during critically low wind speeds under desert conditions. We employed a numerical model to investigate the effect of thickness relative to transpiration, absorptance and leaf size on damage avoidance. We used measured traits and thermotolerance thresholds of real leaves to calculate the leaf temperature response to naturally occurring variable low wind speed. Our results demonstrated that an increase in thickness of only fractions of a millimetre can prevent excursions to damaging high temperatures. This damping effect of increased thickness was greatest when other means of reducing leaf temperature (transpiration, reflectance or reduced size) were lacking. For perennial desert flora, we propose that increased leaf thickness is important in decreasing the incidence of extreme heat stress and, in some species, in enhancing long-term survival.

Wildlife forensics: "supervised" assignment testing can complicate the association of suspect cases to source populations.

Forensic science techniques are an important component of investigations for wildlife-related offences. In particular, DNA analyses can be used to characterize several attributes of biological evidence including sex, individual and species identification. Additionally, genetic assignment testing has enabled forensic biologists to identify the local population from which an individual may have originated. This technique has proved useful in situations where animals have been illegally harvested from areas/populations where hunting is prohibited. For this report, we used individual-based clustering (IBC), in the program Structure 2.2, under both "supervised" and "unsupervised" approaches to assess whether three suspected, illegally harvested moose originated from an endangered population. Atypical circumstances, with Nova Scotia having two moose sub-species in its jurisdiction, enabled strong IBC assignment testing results to determine the source population of the suspected samples. We found differences between the "unsupervised" and "supervised" modeling approaches to define genetic structure among the a priori characterized populations in our data set. Our findings illustrate the fact that individual clustering assignment tests can assist wildlife forensic cases to identify the source population of illegally harvested animals. However, the accuracy of results are highly dependant on the model choice used to define genetic clusters, as well as on the availability of a thorough database of samples throughout the managed area to accurately identify all genetic populations. Further, it is clear from our analyses that political jurisdictions do not accurately reflect isolated populations and we recommend using unsupervised IBC modeling for biological accuracy.

Biocomplexity in mangrove ecosystems.

Mangroves are an ecological assemblage of trees and shrubs adapted to grow in intertidal environments along tropical coasts. Despite repeated demonstration of their economic and societal value, more than 50% of the world's mangroves have been destroyed, 35% in the past two decades to aquaculture and coastal development, altered hydrology, sea-level rise, and nutrient overenrichment. Variations in the structure and function of mangrove ecosystems have generally been described solely on the basis of a hierarchical classification of the physical characteristics of the intertidal environment, including climate, geomorphology, topography, and hydrology. Here, we use the concept of emergent properties at multiple levels within a hierarchical framework to review how the interplay between specialized adaptations and extreme trait plasticity that characterizes mangroves and intertidal environments gives rise to the biocomplexity that distinguishes mangrove ecosystems. The traits that allow mangroves to tolerate variable salinity, flooding, and nutrient availability influence ecosystem processes and ultimately the services they provide. We conclude that an integrated research strategy using emergent properties in empirical and theoretical studies provides a holistic approach for understanding and managing mangrove ecosystems.

The apparent temperature response of leaf respiration depends on the timescale of measurements: a study of two cold climate species.

Productivity and climate models often use a constant Q10 for plant respiration, assuming tight control of respiration by temperature. We studied the temperature response of leaf respiration of two cold climate species (the Australian tree Eucalyptus pauciflora and the subantarctic megaherb Pringlea antiscorbutica, both measured in a field setting) on a short timescale (minutes) during different times within a diel course, and on a longer timescale, using diel variations in ambient temperature. There were great variations in Q10 depending on measuring day, measuring time and measuring method. When Q10 was calculated from short-term (15 min) manipulations of leaf temperature, the resulting values were usually markedly smaller than when Q10 was calculated from measurements at ambient leaf temperatures spread over a day. While for E. pauciflora, Q10 estimates decreased with rising temperature (corroborating the concept of a temperature-dependent Q10), the opposite was the case for P. antiscorbutica. Clearly, factors other than temperature co-regulate both leaf respiration rates and temperature sensitivity and contribute to diel and seasonal variation of respiration.

Testing the growth rate vs. geochemical hypothesis for latitudinal variation in plant nutrients.

Two hypotheses have been proposed to explain increases in plant nitrogen (N) and phosphorus (P) concentrations with latitude: (i) geochemical limitation to P availability in the tropics and (ii) temperature driven variation in growth rate, where greater growth rates (requiring greater nutrient levels) are needed to complete growth and reproduction within shorter growing seasons in temperate than tropical climates. These two hypotheses were assessed in one forest type, intertidal mangroves, using fertilized plots at sites between latitudes 36 masculine S and 27 masculine N. The N and P concentrations in mangrove leaf tissue increased with latitude, but there were no trends in N : P ratios. Growth rates of trees, adjusted for average minimum temperature showed a significant increase with latitude supporting the Growth Rate Hypothesis. However, support for the Geochemical Hypothesis was also strong; both photosynthetic P use efficiency and nutrient resorption efficiency decreased with increasing latitude, indicating that P was less limiting to metabolism at the higher latitudes. Our study supports the hypothesis that historically low P availability in the tropics has been an important selective pressure shaping the evolution of plant traits.

The role of freezing in setting the latitudinal limits of mangrove forests.

Mangrove trees dominate coastal vegetation in tropical regions, but are completely replaced by herbaceous salt marshes at latitudes above 32 degrees N and 40 degrees S. Because water deficit can increase damage caused by freezing, we hypothesized that mangroves, which experience large deficits as a result of saline substrates, would suffer freeze-induced xylem failure. Vulnerability to freeze-induced xylem embolism was examined in the most poleward mangrove species in North America, in an area where freezing is rare but severe, and in Australia, in an area where freezing is frequent but mild. Percentage loss in hydraulic conductivity was measured following manipulations of xylem tension; xylem sap ion concentration was determined using X-ray microanalysis. Species with wider vessels suffered 60-100% loss of hydraulic conductivity after freezing and thawing under tension, while species with narrower vessels lost as little as 13-40% of conductivity. These results indicate that freeze-induced embolism may play a role in setting the latitudinal limits of distribution in mangroves, either through massive embolism following freezing, or through constraints on water transport as a result of vessel size.

Freeze/thaw-induced embolism depends on nadir temperature: the heterogeneous hydration hypothesis.

Freeze/thaw-induced embolism was studied in leaves of field-grown snow gum (Eucalyptus pauciflora) subject to frequent morning frosts. Juvenile trees were grown in buried pots, brought to the laboratory at different stages of acclimation and subjected to simulated frost-freezes (at 2 degrees C h(-1)) to nadir temperatures of -3 or -6 degrees C, which snow gums commonly experience. Frost-frozen and subsequently thawed leaves were cryo-fixed to preserve the distribution of water and were then examined by cryo-scanning electron microscopy. No embolisms were found in leaves frozen to -3 degrees C and thawed. In contrast, 34% of vessels were embolized in thawed leaves that had been frozen to -6 degrees C. This difference was seen also in the extent of extracellular ice blocks in the mid-vein expansion zones in leaves frozen to -3 and -6 degrees C, which occupied 3 and 14% of the mid-vein area, respectively. While the proportion of embolism depended on nadir temperature, it was independent of season (and hence of acclimation state). From the observation that increased embolism at lower nadir temperature was related to the freeze-induced redistribution of water, we hypothesize that the dehydration of cell walls and cells caused by the redistribution exerts sufficient tension on xylem water to induce cavitation on thawing.

A method for assessing occupational exposure to power-frequency magnetic fields for electricity generation and transmission workers.

A new method for assessing both current and historical occupational exposures to magnetic fields has been developed and used in health studies involving a cohort of electricity generation and transmission workers in England and Wales. The exposure values are derived by calculation from engineering and operational data about the power stations rather than from measurements. They are provided for each of 11 job categories for each year of operation of each power station represented in the cohort. The engineering data are used to determine the average magnetic fields in specified areas of work within the power station and then applied to information about the time spent in these areas by each of the job categories. The operational data are used to adjust the exposures for each year according to the power station output for the year. Earlier methods used measurements or the advice of panels of experts to provide exposure scores for a number of job categories across all power stations and years. Such methods were not able to distinguish exposures from different power facilities or during the different years of their operation. Measurement surveys at 10 power stations of the magnetic fields in the work areas gave confidence that the calculations were realistic. Exposure measurements on 215 workers at three power stations were compared in job groups with the exposures predicted by the method. The Pearson correlation coefficient was 0.86 and the slope and intercept of the line of best fit were 0.87 and 0.07 microT respectively. The method gives a good prediction of measured exposure and is being used for studies of occupational exposure to magnetic fields and leukaemia, and of cardiovascular disease, and a reanalysis of brain cancer.

Protection and storage of chlorophyll in overwintering evergreens.

How evergreen species store and protect chlorophyll during exposure to high light in winter remains unexplained. This study reveals that the evergreen snow gum (Eucalyptus pauciflora Sieb. ex Spreng.) stores and protects its chlorophylls by forming special complexes that are unique to the winter-acclimated state. Our in vivo spectral and kinetic characterizations reveal a prominent component of the chlorophyll fluorescence spectrum around 715 nm at 77 K. This band coincides structurally with a loss of chlorophyll and an increase in energy-dissipating carotenoids. Functionally, the band coincides with an increased capacity to dissipate excess light energy, absorbed by the chlorophylls, as heat without intrathylakoid acidification. The increased heat dissipation helps protect the chlorophylls from photo-oxidative bleaching and thereby facilitates rapid recovery of photosynthesis in spring.

A randomized controlled trial of strapping to prevent post-stroke shoulder pain.

OBJECTIVE: To determine whether strapping the shoulder in hemiplegic stroke patients: (1) prevents the development, or reduces the severity, of shoulder pain, (2) preserves range of movement in the shoulder, and (3) improves the functional outcomes for the arm and patient overall. DESIGN: A prospective, randomized, single-blind controlled trial of shoulder strapping versus no strapping. SETTING: Care of the elderly wards in a teaching hospital, Christchurch, New Zealand. SUBJECTS: All patients admitted with an acute hemiplegic stroke, who had persisting weakness of shoulder abduction. INTERVENTION: The treatment group had their affected shoulder strapped for six weeks from randomization in addition to standard physiotherapy. MAIN OUTCOME MEASURES: All subjects were assessed at entry (week 0), at end of the treatment phase (week 6) and two months later (week 14). A visual analogue scale (VAS) was used to assess shoulder pain severity whereas shoulder range of movement to the point of pain (SROMP) assessed passive range of movement and pain. Functional Independence Measure (FIM), Motor Assessment Scale (MAS) and Rankin Disability Index measured functional outcomes. RESULTS: Ninety-eight subjects participated (49 strapped, 49 controls). Intention to treat analysis showed no significant difference in pain, range of movement or functional outcomes after the intervention phase or at the final assessment. However there were trends for less pain at six weeks (VAS, p = 0.11) and better final upper limb function (MAS, p = 0.16) in strapped patients. Skin reactions were uncommon (6.1%). The presence of neglect or sensory loss, but not subluxation, at baseline was independently associated with poor outcome. Range of movement was lost early (mean difference SROMP between hemiplegic and contralateral shoulders at baseline = 15.2 degrees (95% CI 10.9-19.5)) and continued throughout the study. Shoulder strapping did not alter the rate at which range of movement was lost. CONCLUSIONS: No significant benefit with shoulder strapping was demonstrated and reasons for this are discussed. Range of movement in the hemiplegic shoulder is lost very early and any preventive treatments need to begin within the first 1-2 days after a stroke.

Leaf respiration of snow gum in the light and dark. Interactions between temperature and irradiance.

We investigated the effect of temperature and irradiance on leaf respiration (R, non-photorespiratory mitochondrial CO(2) release) of snow gum (Eucalyptus pauciflora Sieb. ex Spreng). Seedlings were hydroponically grown under constant 20 degrees C, controlled-environment conditions. Measurements of R (using the Laisk method) and photosynthesis (at 37 Pa CO(2)) were made at several irradiances (0-2,000 micromol photons m(-2) s(-1)) and temperatures (6 degrees C-30 degrees C). At 15 degrees C to 30 degrees C, substantial inhibition of R occurred at 12 micromol photons m(-2) s(-1), with maximum inhibition occurring at 100 to 200 micromol photons m(-2) s(-1). Higher irradiance had little additional effect on R at these moderate temperatures. The irradiance necessary to maximally inhibit R at 6 degrees C to 10 degrees C was lower than that at 15 degrees C to 30 degrees C. Moreover, although R was inhibited by low irradiance at 6 degrees C to 10 degrees C, it recovered with progressive increases in irradiance. The temperature sensitivity of R was greater in darkness than under bright light. At 30 degrees C and high irradiance, light-inhibited rates of R represented 2% of gross CO(2) uptake (v(c)), whereas photorespiratory CO(2) release was approximately 20% of v(c). If light had not inhibited leaf respiration at 30 degrees C and high irradiance, R would have represented 11% of v(c). Variations in light inhibition of R can therefore have a substantial impact on the proportion of photosynthesis that is respired. We conclude that the rate of R in the light is highly variable, being dependent on irradiance and temperature.

An analysis of falls in the hospital: can we do without bedrails?

OBJECTIVES: To determine the effects of introduction of a bedrail policy, and an educational program, on patient falls and fall-related injuries. DESIGN: A prospective "Before and After" design. PARTICIPANTS AND SETTING: All patients admitted during 1 calendar year in an assessment, treatment, and rehabilitation unit for older people. INTERVENTION: A policy change for the use of bedrails (restricting their use) and an educational program about their effects. MEASUREMENTS: Patient fall rates -- all falls and around the bed falls -- and patient and staff injuries. RESULTS: There was a significant reduction in the number of beds with bedrails attached after the policy introduction (mean of 40/135 vs 18.5/135, respectively, P = .02), but the fall rate (either total or around the bed) did not change significantly. Serious injuries were significantly less common after the bedrail policy was introduced (P = .008), with fewer head injuries. CONCLUSIONS: Reducing the use of bedrails did not alter patient fall rates significantly, but it was associated with a reduction in serious injuries. Unless it can be shown that bedrails are beneficial, their continued use in older patients must be seriously questioned.

The Effect of Elevated [CO2] on Growth and Photosynthesis of Two Eucalyptus Species Exposed to High Temperatures and Water Deficits.

Two species of eucalyptus (Eucalyptus macrorhyncha and Eucalyptus rossii) were grown for 8 weeks in either ambient (350 [mu]L L-1) or elevated (700 [mu]L L-1) CO2 concentrations, either well watered or without water additions, and subjected to a daily, 3-h high-temperature (45[deg]C, maximum) and high-light (1250 [mu]mol photons m-2 s-1, maximum) stress period. Water-stressed seedlings of E. macrorhyncha had higher leaf water potentials when grown in elevated [CO2]. Growth analysis indicated that increased [CO2] may allow eucalyptus species to perform better during conditions of low soil moisture. A down-regulation of photosynthetic capacity was observed for seedlings grown in elevated [CO2] when well watered but not when water stressed. Well-watered seedlings grown in elevated [CO2] had lower quantum efficiencies as measured by chlorophyll fluorescence (the ratio of variable to maximal chlorophyll fluorescence [Fv/Fm]) than seedlings grown in ambient [CO2] during the high-temperature stress period. However, no significant differences in Fv/Fm were observed between CO2 treatments when water was withheld. The reductions in dark-adapted Fv/Fm for plants grown in elevated [CO2] were not well correlated with increased xanthophyll cycle photoprotection. However, reductions in the Fv/Fm were correlated with increased levels of nonstructural carbohydrates. The reduction in quantum efficiencies for plants grown in elevated [CO2] is discussed in the context of feedback inhibition of electron transport associated with starch accumulation and variation in sink strength.

Transfer of PCDD/PCDF from contaminated soils into carrots, lettuce and peas.

In a field experiment, the PCDD/PCDF transfer pathways from soil into carrots, lettuce and peas has been investigated. PCDD/PCDF contamination levels in soil varied between 5 ng I-TEq/kg on the control plot and 56 ng I-TEq/kg on the contaminated plot. PCDD/PCDF levels in carrots were threefold higher in the contaminated plot than in the control plot, which was a result of a tenfold increase in the PCDD/PCDF levels of the peel. PCDD/PCDF levels in lettuce and peas were not higher when grown on the contaminated plot and were much lower than in carrots, which indicates that the PCDD/PCDF in lettuce and peas from both plots are of atmospheric origin.

Dimerization of 2,6-dimethoxyphenol by Aspergillus flavus: evidence for the reaction occurring close to mycelia.

Aspergillus flavus grown on 2,6-dimethoxyphenol as sole carbon source produced tetramethoxy-p-dibenzoquinone by a free radical mechanism. The product was identified by H-nmr and ms. Scanning electron microscopy and light microscopy were used to follow the growth of mycelia and the attachment of crystals to the mycelial surfaces. Formation of dimer was inhibited by the presence of glucose in the medium.