Automated robotic harvesting of protein crystals—addressing a critical bottleneck or instrumentation overkill?

One of the critical steps in high throughput crystallography that so far has evaded automation is the actual harvesting of the delicate crystals from the mother liquor in which they are growing. The late-stage operation of harvesting is presently a most risky and loss-intensive procedure, compounded by its tight integration with the critical steps of cryo-protection and cryo-quenching. Recent advances in micromanipulation robotics and micro-fabrication have made it possible to seriously consider automation of protein crystal harvesting. Based on the experience gained during the development of an operator-assisted (and now operator-assisting) universal micromanipulation robot (UMR) prototype, we discuss the challenges ahead for the design of a fully autonomous, integrated system capable of the reliable harvesting of protein microcrystals. Experience from participation in NIH structural genomics projects and feedback from bottleneck workshops indicates that genuine demand exists in the high throughput community as well as in pharmaceutical production pipelines, justifying the effort and resources to develop autonomous harvesting robotics.     

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

ATGL and HSL are not coordinately regulated in response to fuel partitioning in fasted rats

Prolonged fasting is characterized by lipid mobilization (Phase 2), followed by protein breakdown (Phase 3). Knowing that body lipids are not exhausted in Phase 3, we investigated whether changes in the metabolic status of prolonged fasted rats are associated with differences in the expression of epididymal adipose tissue proteins involved in lipid mobilization. The final body mass, body lipid content, locomotor activity and metabolite and hormone plasma levels differed between groups. Compared with fed rats, adiposity and epididymal fat mass decreased in Phase 2 (approximately two- to threefold) and Phase 3 (∼4.5-14-fold). Plasma nonesterified fatty acids (NEFA) concentrations were increased in Phase 2 (approximately twofold) and decreased in Phase 3 (approximately twofold). Daily locomotor activity was markedly increased in Phase 3 (∼11-fold). Compared with the fed state, expressions of adipose triglyceride lipase (ATGL; mRNA and protein), hormone-sensitive lipase (HSL; mRNA) and phosphorylated HSL at residue Ser660 (HSL Ser⁶⁶⁰) were increased during Phase 2 (∼1.5-2-fold). HSL (mRNA and protein) and HSL Ser⁶⁶⁰ levels were lowered during Phase 3 (∼3-12-fold). Unlike HSL and HSL Ser⁶⁶⁰, ATGL expression did not correlate with circulating NEFA, mostly due to data from animals in Phase 3. At this stage, ATGL could play an essential role for maintaining a low mobilization rate of NEFA, possibly to sustain muscle performance and hence increased locomotor activity. We conclude that ATGL and HSL are not coordinately regulated in response to changes in fuel partitioning during prolonged food deprivation, ATGL appearing as the major lipase in late fasting.     

Performance evaluation of native plants suited to extensive green roof conditions in a maritime climate

Assessing plant species performance on extensive green roofs can inform about and improve green roof functioning, aesthetics, longevity and the diversity of plant palettes available for the green roof industry. In this study, we evaluate survival, cover, roof cooling and stormwater retention properties of 15 plant species native to coastal regions of Atlantic Canada in extensive green roof monocultures. After a complete growing season (May-October 2009), all but one species had greater than 80% survival, and 10 species reached greater than 90% groundcover. Over the growing season, the top performing species reduced roof surface temperature by an average of 3.44 °C and increased solar reflectivity by 22.2% over the growing-medium only controls. Moreover, the best species retained 75.3% of experimentally added stormwater. Our results demonstrate that several species (mainly graminoids) performed better than creeping shrubs and forbs for most functions, although significant variation existed within life-form groups.     

Electrophysiological identification of cold receptors on the antennae of the ground beetle Pterostichus aethiops

Abstract.  In single-sensillum extracellular electrophysiological recordings, terminal campaniform sensilla at the tip of antennae of the ground beetle Pterostichus aethiops (Pz., 1797) show action potentials of three sensory cells, A-, B- and C-cells, distinguished by differences in their spike amplitudes. Only the A-cell, with the largest spike amplitude, is highly sensitive to temperature fluctuations, showing remarkable changes in its firing rate induced by changes in temperature of 0.1 °C. The firing rate of A-cells at 23 °C varies from 15–52 Hz among different beetles. Mean impulse frequency of A-cells is found to be a function of steady temperature, the firing rate decreasing with temperature increase. A-cells respond to a rapid temperature drop with a strong phasic-tonic reaction; larger decreases in temperature evoke higher peak frequency values. Maximum peak frequencies, varying from 380–630 Hz in different beetles, are induced by temperature decreases of 3–10 °C, whereas temperature rise strongly inhibits impulse activity of the A-cell. The first manifestation of rapid warming in the nerve impulse sequence is a very long interspike period, followed by diminished activity. Both the length of the long interspike period and the rate of following impulse activity are functions of temperature change; hence, A-cells respond to temperature changes as typical cold receptors, similar to coeloconic and short hair-like sensilla in other insects.     

Fire‐induced tree mortality in a neotropical forest: the roles of bark traits,tree size,wood density and fire behavior

Large‐scale wildfires are expected to accelerate forest dieback in Amazônia, but the fire vulnerability of tree species remains uncertain, in part due to the lack of studies relating fire‐induced mortality to both fire behavior and plant traits. To address this gap, we established two sets of experiments in southern Amazonia. First, we tested which bark traits best predict heat transfer rates (R) through bark during experimental bole heating. Second, using data from a large‐scale fire experiment, we tested the effects of tree wood density (WD), size, and estimated R (inverse of cambium insulation) on tree mortality after one to five fires. In the first experiment, bark thickness explained 82% of the variance in R, while the presence of water in the bark reduced the difference in temperature between the heat source and the vascular cambium, perhaps because of high latent heat of vaporization. This novel finding provides an important insight for improving mechanistic models of fire‐induced cambium damage from tropical to temperate regions. In the second experiment, tree mortality increased with increasing fire intensity (i.e. as indicated by bark char height on tree boles), which was higher along the forest edge, during the 2007 drought, and when the fire return interval was 3 years instead of one. Contrary to other tropical studies, the relationship between mortality and fire intensity was strongest in the year following the fires, but continued for 3 years afterwards. Tree mortality was low (≤20%) for thick‐barked individuals (≥18 mm) subjected to medium‐intensity fires, and significantly decreased as a function of increasing tree diameter, height and wood density. Hence, fire‐induced tree mortality was influenced not only by cambium insulation but also by other traits that reduce the indirect effects of fire. These results can be used to improve assessments of fire vulnerability of tropical forests.     

Materials Challenges for High Performance Magnetocaloric Refrigeration Devices

Magnetocaloric materials with a Curie temperature near room temperature have attracted significant interest for some time due to their possible application for high‐efficiency refrigeration devices. This review focuses on a number of key issues of relevance for the characterization, performance and implementation of such materials in actual devices. The phenomenology and fundamental thermodynamics of magnetocaloric materials is discussed, as well as the hysteresis behavior often found in first‐order materials. A number of theoretical and experimental approaches and their implications are reviewed. The question of how to evaluate the suitability of a given material for use in a magnetocaloric device is covered in some detail, including a critical assessment of a number of common performance metrics. Of particular interest is which non‐magnetocaloric properties need to be considered in this connection. An overview of several important materials classes is given before considering the performance of materials in actual devices. Finally, an outlook on further developments is presented.     

Brain cooling maintenance with cooling cap following induction with intracarotid cold saline infusion: a quantitative model

Intracarotid cold saline infusion (ICSI) is potentially much faster than whole-body cooling and more effective than cooling caps in inducing therapeutic brain cooling. One drawback of ICSI is hemodilution and volume loading. We hypothesized that cooling caps could enhance brain cooling with ICSI and minimize hemodilution and volume loading. Six-hour-long simulations were performed in a 3D mathematical brain model. The Pennes bioheat equation was used to propagate brain temperature. Convective heat transfer through jugular venous return and the circle of Willis was simulated. Hemodilution and volume loading were modeled using a two-compartment saline infusion model. A feedback method of local brain temperature control was developed where ICSI flow rate was varied based on the rate of temperature change and the deviation of temperature to a target (32 °C) within a voxel in the treated region of brain. The simulations confirmed the inability of cooling caps alone to induce hypothermia. In the ICSI and the combination models (ICSI and cap), the control algorithm guided ICSI to quickly achieve and maintain the target temperature. The combination model had lower ICSI flow rates than the ICSI model resulting in a 55% reduction of infusion volume over a 6 h period and higher hematocrit values compared to the ICSI model. Moreover, in the combination model, the ICSI flow rate decreased to zero after 4 h, and hypothermia was subsequently maintained solely by the cooling cap. This is the first study supporting a role of cooling caps in therapeutic hypothermia in adults.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.