Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

The roles of conduit redundancy and connectivity in xylem hydraulic functions.

Wood anatomical traits shape a xylem segment's hydraulic efficiency and resistance to embolism spread due to declining water potential. It has been known for decades that variations in conduit connectivity play a role in altering xylem hydraulics. However, evaluating the precise effect of conduit connectivity has been elusive. The objective here is to establish an analytical linkage between conduit connectivity and grouping and tissue-scale hydraulics. It is hypothesized that an increase in conduit connectivity brings improved resistance to embolism spread due to increased hydraulic pathway redundancy. However, an increase in conduit connectivity could also reduce resistance due to increased speed of embolism spread with respect to pressure. We elaborate on this trade-off using graph theory, percolation theory and computational modeling of xylem. The results are validated using anatomical measurements of Acer branch xylem. Considering only species with vessels, increases in connectivity improve resistance to embolism spread without negatively affecting hydraulic conductivity. The often measured grouping index fails to capture the totality of the effect of conduit connectivity on xylem hydraulics. Variations in xylem network characteristics, such as conduit connectivity, might explain why hypothesized trends among woody species, such as the 'safety-efficiency' trade-off hypothesis, are weaker than expected.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Visualizing Magnetic Structure in 3D Nanoscale Ni-Fe Gyroid Networks.

Arrays of interacting 2D nanomagnets display unprecedented electromagnetic properties via collective effects, demonstrated in artificial spin ices and magnonic crystals. Progress toward 3D magnetic metamaterials is hampered by two challenges: fabricating 3D structures near intrinsic magnetic length scales (sub-100 nm) and visualizing their magnetic configurations. Here, we fabricate and measure nanoscale magnetic gyroids, periodic chiral networks comprising nanowire-like struts forming three-connected vertices. Via block copolymer templating, we produce Ni75Fe25 single-gyroid and double-gyroid (an inversion pair of single-gyroids) nanostructures with a 42 nm unit cell and 11 nm diameter struts, comparable to the exchange length in Ni-Fe. We visualize their magnetization distributions via off-axis electron holography with nanometer spatial resolution and interpret the patterns using finite-element micromagnetic simulations. Our results suggest an intricate, frustrated remanent state which is ferromagnetic but without a unique equilibrium configuration, opening new possibilities for collective phenomena in magnetism, including 3D magnonic crystals and unconventional computing.

PERINATAL CENTRILOBULAR HEPATIC NECROSIS IN GIANT PANDAS (AILUROPODA MELANOLEUCA): A RETROSPECTIVE STUDY.

Between 1983 and 2012, six giant panda cubs (Ailuropoda melanoleuca) born at a zoological institution were stillborn or died between the ages of 3 and 200 h. Two of the six cubs had panhepatic centrilobular hepatic necrosis (CHN), granulocytic extramedullary hematopoiesis (GEM), positive liver culture for Staphylococcus species, and terminal liver failure. Another low-weight cub was administered oxygen therapy immediately after birth and developed hyaline membranes in air spaces and hepatic necrosis restricted to the hilar region. A retrospective analysis of liver and lung lesions, pulmonary microanatomy, blood-gas barrier ultrastructure, and hepatic myofibroblast proliferation was conducted on the six cubs. Neonates with CHN had concurrent severe periportal GEM accompanied by severe myofibroblast proliferation. The pulmonary blood-gas barrier was markedly increased in one cub with CHN. Developmentally, the lungs of all but one cub were at the late saccular stage, and the lowest-weight cub was in early saccular stage, consistent with immaturity, and had pneumonia comparable to neonatal respiratory distress syndrome (RDS). Stage of lung development was eliminated as the primary factor leading to CHN. The pathogenesis of CHN in these neonates is proposed to be transformation of hepatic stellate cells to myofibroblasts initiating blockage and microvascular constriction of hepatic sinusoids, resulting in insufficient perfusion and cellular hypoxia of hepatocytes surrounding central veins in acinar zone 3.

TUBERCULOSIS CAUSED BY MYCOBACTERIUM ORYGIS IN A GREATER ONE-HORNED RHINOCEROS (RHINOCEROS UNICORNIS): FIRST REPORT IN THE WESTERN HEMISPHERE.

Mycobacterium orygis, a newly identified member of the Mycobacterium tuberculosis complex, has been isolated predominantly from hoofstock in eastern Africa and the Arabian Peninsula, and sporadically in cattle (Bos taurus indicus), rhesus monkeys (Macaca mulatta), humans, and a greater one-horned rhinoceros (Rhinoceros unicornis) in South Asia. In rhinoceros, tuberculosis typically presents as a chronic progressive respiratory disease. The report describes the postmortem diagnosis of tuberculosis caused by Mycobacterium orygis in a greater one-horned rhinoceros with hind limb paresis due to neural granulomatosis. Serologic assays for detection of antibodies to M. tuberculosis complex proteins before culture results allowed for appropriate herd management protocols to be initiated. Mycobacterium genus-specific polymerase chain reaction assays with direct sequencing allowed timely confirmation of the serologic results. This is the first isolation of M. orygis in the western hemisphere, showing the need for mycobacterial testing of rhinoceros before international shipments and the urgency for validated antemortem M. tuberculosis complex screening assays in rhinoceros species.

DERMATITIS AND RHINOSINUITIS CAUSED BY CURVULARIA SPECIES IN A CHINESE GORAL (NAEMORHEDUS GRISEUS).

Curvularia spp. are globally distributed saprophytic fungi, classified in the literature as dematiaceous, or darkly pigmented fungi. These fungi have been increasingly recognized as causing cutaneous, ocular, respiratory, and central nervous system infections in humans, but have been infrequently documented as pathogens in the veterinary literature. A 5-yr-old male Chinese goral (Naemorhedus griseus) presented with bilateral fungal dermatitis of the pinnae, and subsequent pyogranulomatous rhinosinusitis. Clinical signs included epistaxis, mucosanguineous nasal discharge, and dyspnea. Sequential histologic examinations of cutaneous and nasal lesions revealed pyogranulomatous inflammation with extracellular and phagocytized nonpigmented yeasts. Fungal culture and polymerase chain reaction identified Curvularia sp. The absence of pigmentation in tissue in this case suggests that pigmentation may not be a consistent histologic finding for this fungus, emphasizing the importance of molecular identification to prevent misidentification. Despite intensive interventions in this goral, the disease progressed, and was ultimately fatal.

A stomatal control model based on optimization of carbon gain versus hydraulic risk predicts aspen sapling responses to drought.

Empirical models of plant drought responses rely on parameters that are difficult to specify a priori. We test a trait- and process-based model to predict environmental responses from an optimization of carbon gain vs hydraulic risk. We applied four drought treatments to aspen (Populus tremuloides) saplings in a research garden. First we tested the optimization algorithm by using predawn xylem pressure as an input. We then tested the full model which calculates root-zone water budget and xylem pressure hourly throughout the growing season. The optimization algorithm performed well when run from measured predawn pressures. The per cent mean absolute error (MAE) averaged 27.7% for midday xylem pressure, transpiration, net assimilation, leaf temperature, sapflow, diffusive conductance and soil-canopy hydraulic conductance. Average MAE was 31.2% for the same observations when the full model was run from irrigation and rain data. Saplings that died were projected to exceed 85% loss in soil-canopy hydraulic conductance, whereas surviving plants never reached this threshold. The model fit was equivalent to that of an empirical model, but with the advantage that all inputs are specific traits. Prediction is empowered because knowing these traits allows knowing the response to climatic stress.

Predicting stomatal responses to the environment from the optimization of photosynthetic gain and hydraulic cost.

Stomatal regulation presumably evolved to optimize CO2 for H2 O exchange in response to changing conditions. If the optimization criterion can be readily measured or calculated, then stomatal responses can be efficiently modelled without recourse to empirical models or underlying mechanism. Previous efforts have been challenged by the lack of a transparent index for the cost of losing water. Yet it is accepted that stomata control water loss to avoid excessive loss of hydraulic conductance from cavitation and soil drying. Proximity to hydraulic failure and desiccation can represent the cost of water loss. If at any given instant, the stomatal aperture adjusts to maximize the instantaneous difference between photosynthetic gain and hydraulic cost, then a model can predict the trajectory of stomatal responses to changes in environment across time. Results of this optimization model are consistent with the widely used Ball-Berry-Leuning empirical model (r2 > 0.99) across a wide range of vapour pressure deficits and ambient CO2 concentrations for wet soil. The advantage of the optimization approach is the absence of empirical coefficients, applicability to dry as well as wet soil and prediction of plant hydraulic status along with gas exchange.

What plant hydraulics can tell us about responses to climate-change droughts.

Climate change exposes vegetation to unusual drought, causing declines in productivity and increased mortality. Drought responses are hard to anticipate because canopy transpiration and diffusive conductance (G) respond to drying soil and vapor pressure deficit (D) in complex ways. A growing database of hydraulic traits, combined with a parsimonious theory of tree water transport and its regulation, may improve predictions of at-risk vegetation. The theory uses the physics of flow through soil and xylem to quantify how canopy water supply declines with drought and ceases by hydraulic failure. This transpiration 'supply function' is used to predict a water 'loss function' by assuming that stomatal regulation exploits transport capacity while avoiding failure. Supply-loss theory incorporates root distribution, hydraulic redistribution, cavitation vulnerability, and cavitation reversal. The theory efficiently defines stomatal responses to D, drying soil, and hydraulic vulnerability. Driving the theory with climate predicts drought-induced loss of plant hydraulic conductance (k), canopy G, carbon assimilation, and productivity. Data lead to the 'chronic stress hypothesis' wherein > 60% loss of k increases mortality by multiple mechanisms. Supply-loss theory predicts the climatic conditions that push vegetation over this risk threshold. The theory's simplicity and predictive power encourage testing and application in large-scale modeling.

Determining the Proximity Effect-Induced Magnetic Moment in Graphene by Polarized Neutron Reflectivity and X-ray Magnetic Circular Dichroism.

We report the magnitude of the induced magnetic moment in CVD-grown epitaxial and rotated-domain graphene in proximity with a ferromagnetic Ni film, using polarized neutron reflectivity (PNR) and X-ray magnetic circular dichroism (XMCD). The XMCD spectra at the C K-edge confirm the presence of a magnetic signal in the graphene layer, and the sum rules give a magnetic moment of up to ∼0.47 µB/C atom induced in the graphene layer. For a more precise estimation, we conducted PNR measurements. The PNR results indicate an induced magnetic moment of ∼0.41 µB/C atom at 10 K for epitaxial and rotated-domain graphene. Additional PNR measurements on graphene grown on a nonmagnetic Ni9Mo1 substrate, where no magnetic moment in graphene is measured, suggest that the origin of the induced magnetic moment is due to the opening of the graphene's Dirac cone as a result of the strong C pz-Ni 3d hybridization.

'Pressure fatigue': the influence of sap pressure cycles on cavitation vulnerability in Acer negundo.

Vulnerability-to-cavitation curves (VCs) can vary within a tree crown in relation to position or branch age. We tested the hypothesis that VC variation can arise from differential susceptibility to the number of diurnal sap pressure cycles experienced. We designed a method to distinguish between effects of cycling vs exposure time to negative pressure, and tested the influence of sap pressure cycles on cavitation vulnerability between upper and lower branches in Acer negundo L. trees using static and flow centrifuge, and air-injection methods. Branches from the upper crown had greater hydraulic conductivity and were more resistant to cavitation than branches from the lower crown. Upper branches also showed little change after exposure to 10 or 20 pressure cycles between -0.5 MPa and -2.0 MPa. Lower branches, however, showed a marked increase in vulnerability to cavitation after pressure-cycling. This result suggests that 'cavitation fatigue' can occur without the actual induction (and reversal) of cavitation as documented previously, but simply from the cycling of pressures in the sub-cavitation range. This 'pressure fatigue' may explain age-related shifts in VCs that could eventually induce dieback in suppressed branches or trees. Pressure fatigue may help explain developmental variation in hydraulic capacity of branches within individuals.

The stomatal response to rising CO2 concentration and drought is predicted by a hydraulic trait-based optimization model.

Modeling stomatal control is critical for predicting forest responses to the changing environment and hence the global water and carbon cycles. A trait-based stomatal control model that optimizes carbon gain while avoiding hydraulic risk has been shown to perform well in response to drought. However, the model's performance against changes in atmospheric CO2, which is rising rapidly due to human emissions, has yet to be evaluated. The present study tested the gain-risk model's ability to predict the stomatal response to CO2 concentration with potted water birch (Betula occidentalis Hook.) saplings in a growth chamber. The model's performance in predicting stomatal response to changes in atmospheric relative humidity and soil moisture was also assessed. The gain-risk model predicted the photosynthetic assimilation, transpiration rate and leaf xylem pressure under different CO2 concentrations, having a mean absolute percentage error (MAPE) of 25%. The model also predicted the responses to relative humidity and soil drought with a MAPE of 21.9% and 41.9%, respectively. Overall, the gain-risk model had an MAPE of 26.8% compared with the 37.5% MAPE obtained by a standard empirical model of stomatal conductance. Importantly, unlike empirical models, the optimization model relies on measurable physiological traits as inputs and performs well in predicting responses to novel environmental conditions without empirical corrections. Incorporating the optimization model in larger scale models has the potential for improving the simulation of water and carbon cycles.

In situ embolism induction reveals vessel refilling in a natural aspen stand.

Little is known about the ability of trees to recover hydraulic conductance (k) within a growing season by regrowth or refilling of embolized conduits. Recovery of k lost to drought or other causes would prevent chronic reductions in gas exchange and productivity. To test recovery ability we conducted a 2-year experiment (2014-15) on a cohort of aspen ramets (Populus tremuloides, Michx.). Whole-tree k was measured from mid-June through September from sapflow (Q) and pre-dawn and mid-day xylem pressure. We induced embolism in the treatment group with high air pressure delivered by a split pressure chamber sealed around the main trunk. Successful treatments reduced k and Q by 50% or more without causing rapid desiccation. The majority of trees recovered following treatment, rising to control levels of k and Q between 12 and 17 days. Failure to recover was correlated with drier climate conditions. The growing-season recovery of k was attributed to refilling of embolized vessels, based on the absence of diameter growth. Pre-dawn xylem pressures during recovery were similar to the threshold needed to passively collapse emboli. Successful recovery during the 2-year study was consistent with no reduction in cumulative Q or canopy area in treatment vs controls. However, non-recovering trees in 2014 exhibited lower basal area growth at the start of the 2015 growing season, suggesting a linkage between recovery ability and productivity. This study provides evidence for the potential of trees to recover xylem function by refilling during the growing season.

A multi-species synthesis of physiological mechanisms in drought-induced tree mortality.

Widespread tree mortality associated with drought has been observed on all forested continents and global change is expected to exacerbate vegetation vulnerability. Forest mortality has implications for future biosphere-atmosphere interactions of carbon, water and energy balance, and is poorly represented in dynamic vegetation models. Reducing uncertainty requires improved mortality projections founded on robust physiological processes. However, the proposed mechanisms of drought-induced mortality, including hydraulic failure and carbon starvation, are unresolved. A growing number of empirical studies have investigated these mechanisms, but data have not been consistently analysed across species and biomes using a standardized physiological framework. Here, we show that xylem hydraulic failure was ubiquitous across multiple tree taxa at drought-induced mortality. All species assessed had 60% or higher loss of xylem hydraulic conductivity, consistent with proposed theoretical and modelled survival thresholds. We found diverse responses in non-structural carbohydrate reserves at mortality, indicating that evidence supporting carbon starvation was not universal. Reduced non-structural carbohydrates were more common for gymnosperms than angiosperms, associated with xylem hydraulic vulnerability, and may have a role in reducing hydraulic function. Our finding that hydraulic failure at drought-induced mortality was persistent across species indicates that substantial improvement in vegetation modelling can be achieved using thresholds in hydraulic function.

The Scanning TMR Microscope for Biosensor Applications.

We present a novel tunnel magnetoresistance (TMR) scanning microscope set-up capable of quantitatively imaging the magnetic stray field patterns of micron-sized elements in 3D. By incorporating an Anderson loop measurement circuit for impedance matching, we are able to detect magnetoresistance changes of as little as 0.006%/Oe. By 3D rastering a mounted TMR sensor over our magnetic barcodes, we are able to characterize the complex domain structures by displaying the real component, the amplitude and the phase of the sensor's impedance. The modular design, incorporating a TMR sensor with an optical microscope, renders this set-up a versatile platform for studying and imaging immobilised magnetic carriers and barcodes currently employed in biosensor platforms, magnetotactic bacteria and other complex magnetic domain structures of micron-sized entities. The quantitative nature of the instrument and its ability to produce vector maps of magnetic stray fields has the potential to provide significant advantages over other commonly used scanning magnetometry techniques.

Magnetically labelled gold and epoxy bi-functional microcarriers for suspension based bioassay technologies.

Microarrays and suspension-based assay technologies have attracted significant interest over the past decade with applications ranging from medical diagnostics to high throughput molecular biology. The throughput and sensitivity of a microarray will always be limited by the array density and slow reaction kinetics. Suspension (or bead) based technologies offer a conceptually different approach, improving detection by substituting a fixed plane of operation with many individually distinguishable microcarriers. In addition to all the features of a suspension based assay technology, our technology offers a rewritable label. This has the potential to be truly revolutionary by opening up the possibility of generating, on chip, extensive labelled molecular libraries. We unveil our latest SU-8 microcarrier design with embedded magnetic films that can be utilized for both magnetic and optical labelling. The novel design significantly simplifies fabrication and additionally incorporates a gold cap to provide a dual surface, bi-functional architecture. The microcarriers are fabricated using deep-ultraviolet lithography techniques and metallic thin film growth by evaporation. The bi-functional properties of the microcarriers will allow us to use each microcarrier as its own positive control thereby increasing the reliability of our technology. Here we present details of the design, fabrication, magnetic detection and functionalization of these microcarriers.