Variable temperature effects of Open Top Chambers at polar and alpine sites explained by irradiance and snow depth

Environmental manipulation studies are integral to determining biological consequences of climate warming. Open Top Chambers (OTCs) have been widely used to assess summer warming effects on terrestrial biota, with their effects during other seasons normally being given less attention even though chambers are often deployed year‐round. In addition, their effects on temperature extremes and freeze‐thaw events are poorly documented. To provide robust documentation of the microclimatic influences of OTCs throughout the year, we analysed temperature data from 20 studies distributed across polar and alpine regions. The effects of OTCs on mean temperature showed a large range (?0.9 to 2.1 °C) throughout the year, but did not differ significantly between studies. Increases in mean monthly and diurnal temperature were strongly related (R² = 0.70) with irradiance, indicating that PAR can be used to predict the mean warming effect of OTCs. Deeper snow trapped in OTCs also induced higher temperatures at soil/vegetation level. OTC‐induced changes in the frequency of freeze‐thaw events included an increase in autumn and decreases in spring and summer. Frequency of high‐temperature events in OTCs increased in spring, summer and autumn compared with non‐manipulated control plots. Frequency of low‐temperature events was reduced by deeper snow accumulation and higher mean temperatures. The strong interactions identified between aspects of ambient environmental conditions and effects of OTCs suggest that a detailed knowledge of snow depth, temperature and irradiance levels enables us to predict how OTCs will modify the microclimate at a particular site and season. Such predictive power allows a better mechanistic understanding of observed biotic response to experimental warming studies and for more informed design of future experiments. However, a need remains to quantify OTC effects on water availability and wind speed (affecting, for example, drying rates and water stress) in combination with microclimate measurements at organism level.     

Dynamic and complex microclimate responses to warming and grazing manipulations

Synthesis efforts that identify patterns of ecosystem response to a suite of warming manipulations can make important contributions to climate change science. However, cross‐study comparisons are impeded by the paucity of detailed analyses of how passive warming and other manipulations affect microclimate. Here we document the independent and combined effects of a common passive warming manipulation, open‐top chambers (OTCs), and a simulated widespread land use, clipping, on microclimate on the Tibetan Plateau. OTCs consistently elevated growing season averaged mean daily air temperature by 1.0–2.0°C, maximum daily air temperature by 2.1–7.3°C and the diurnal air temperature range by 1.9–6.5°C, with mixed effects on minimum daily air temperature, and mean daily soil temperature and moisture. These OTC effects on microclimate differ from reported effects of a common active warming method, infrared heating, which has more consistent effects on soil than on air temperature. There were significant interannual and intragrowing season differences in OTC effects on microclimate. For example, while OTCs had mixed effects on growing season averaged soil temperatures, OTCs consistently elevated soil temperature by approximately 1.0°C early in the growing season. Nonadditive interactions between OTCs and clipping were also present: OTCs in clipped plots generally elevated air and soil temperatures more than OTCs in nonclipped plots. Moreover, site factors dynamically interacted with microclimate and with the efficacy of the OTC manipulations. These findings highlight the need to understand differential microclimate effects between warming methods, within warming method across ecosystem sites, within warming method crossed with other treatments, and within sites over various timescales. Methods, sites and scales are potential explanatory variables and covariables in climate warming experiments. Consideration of this variability among and between experimental warming studies will lead to greater understanding and better prediction of ecosystem response to anthropogenic climate warming.     

Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.     

Mild experimental climate warming induces metabolic impairment and massive mortalities in southern African quartz field succulents

Current thermal regimes for many southern African succulent species of the subfamily Ruschioideae, which rapidly diversified during the cooler Pleistocene period, may be close to their tolerable extremes, and likely exceeded with anticipated future climate warming. This hypothesis was tested by exposing succulent species of different size and architecture to differently elevated temperatures approximating future African climate scenarios (2.5–3.8 °C increases in mean annual daily temperature maxima) using transparent hexagonal open-top chambers of different heights. Air temperatures, soil water potentials and amounts of fog and dew precipitation were monitored hourly in the differently heated open-top chambers and ambient environment, and changes in species leaf densities and canopy covers precisely determined in these chambers and ambient environment from high resolution digital images taken at 3-monthly intervals spanning a 12-month monitoring period. Photochemical efficiencies and activities of the photosynthetic enzyme Rubisco were also measured in one widespread dwarf succulent species following 2-h exposures of its populations in a forced draft oven to eight different heat intensities (range: 40–54 °C), the highest closely matching the temperature extreme of 54.8 °C recorded in the most intensively heated open-top chambers. After 12-months warming, all succulent species displayed massively (up to 90.2%) reduced leaf densities and canopy covers in the differently heated open-top chambers, with small sparsely branched species comprising single leaf pairs per axis exhibiting much greater reductions than large, shrubby or creeping species with multiple leaves. Noteworthy, was that fog and dew precipitation levels and soil water potentials at the centres of the least intensively heated chambers did not differ significantly from those in the ambient environment, even during the critical dry summer and early autumn seasons. However, leaf density and canopy reductions in these chambers were of similar magnitude to those in the most intensively heated chambers where fog and dew precipitation levels and soil water potentials were significantly reduced. These findings identified elevated temperatures as the principal cause of the observed massive reductions in leaf density and canopy cover and supported the hypothesis that mild anthropogenic warming could exceed the thermal thresholds of many southern African quartz field succulents leading to metabolic impairment. This impairment explained by an observed loss in the catalytic efficiency of Rubisco at daytime temperature extremes exceeding 54 °C, preceded by a decrease in PSII electron transport commencing at temperatures much lower than the threshold for Rubisco de-activation.     

Short-term effects of simulated environmental change on phenology, leaf traits, and shoot growth of alpine plants on a temperate mountain, northern Japan

In order to assess the responses of circumpolar and semicircumpolar plants growing around their southern distribution margins to artificial warming, we set up 11 open-top chambers (OTCs) on a fell-field (1680 m a.s.l.) in the Taisetsu Mountains, northern Japan. The OTCs increased mean air temperature by 1.3°C through the growing season (June–September) and extended the length of the growing season. We examined phenology and leaf traits of plants in the OTCs and control plots during the first season under artificial warming treatment using two deciduous and three evergreen species. Ledum palustre (evergreen shrub), Vaccinium uliginosum , and Arctous alpinus (deciduous shrubs) showed earlier leaf emergence and/or flowering in the OTCs. Deciduous shrubs had longer individual leaf longevity and an extended foliage period in the OTCs than in the control plots. There were no significant differences in specific leaf area and leaf size for many species between the OTCs and the control plots. Vaccinium vitis-idaea (evergreen shrub), L. palustre, A. alpinus , and Empetrum nigrum (evergreen shrub) had lower leaf nitrogen concentration in the OTCs than in the control plots, whereas it was higher in V. uliginosum . Only E. nigrum showed larger annual shoot growth in the OTCs. No clear differences in response to the warming effect were detected between evergreen and deciduous species in the first season. Circumpolar plants growing in temperate alpine regions may be more affected by season length rather than temperature itself.     

Leaf photosynthesis and simulated carbon budget of Gentiana straminea from a decade-long warming experiment

Aims Alpine ecosystems may experience larger temperature increases due to global warming as compared with lowland ecosystems. Information on physiological adjustment of alpine plants to temperature changes can provide insights into our understanding how these plants are responding to current and future warming. We tested the hypothesis that alpine plants would exhibit acclimation in photosynthesis and respiration under long-term elevated temperature, and the acclimation may relatively increase leaf carbon gain under warming conditions.Methods Open-top chambers (OTCs) were set up for a period of 11 years to artificially increase the temperature in an alpine meadow ecosystem. We measured leaf photosynthesis and dark respiration under different light, temperature and ambient CO₂ concentrations for Gentiana straminea, a species widely distributed on the Tibetan Plateau. Maximum rates of the photosynthetic electron transport (J max), RuBP carboxylation (V c max) and temperature sensitivity of respiration Q 10 were obtained from the measurements. We further estimated the leaf carbon budget of G. straminea using the physiological parameters and environmental variables obtained in the study.Important findings1)?The OTCs consistently elevated the daily mean air temperature by ~1.6°C and soil temperature by ~0.5°C during the growing season. 2)?Despite the small difference in the temperature environment, there was strong tendency in the temperature acclimation of photosynthesis. The estimated temperature optimum of light-saturated photosynthetic CO₂ uptake (A max) shifted ~1°C higher from the plants under the ambient regime to those under the OTCs warming regime, and the A max was significantly lower in the warming-acclimated leaves than the leaves outside the OTCs. 3)?Temperature acclimation of respiration was large and significant: the dark respiration rates of leaves developed in the warming regime were significantly lower than leaves from the ambient environments. 4)?The simulated net leaf carbon gain was significantly lower in the in situ leaves under the OTCs warming regime than under the ambient open regime. However, in comparison with the assumed non-acclimation leaves, the in situ warming-acclimated leaves exhibited significantly higher net leaf carbon gain. 5)?The results suggest that there was a strong and significant temperature acclimation in physiology of G. straminea in response to long-term warming, and the physiological acclimation can reduce the decrease of leaf carbon gain, i.e. increase relatively leaf carbon gain under the warming condition in the alpine species.     

Effects of experimental warming of air,soil and permafrost on carbon balance in Alaskan tundra

The carbon (C) storage capacity of northern latitude ecosystems may diminish as warming air temperatures increase permafrost thaw and stimulate decomposition of previously frozen soil organic C. However, warming may also enhance plant growth so that photosynthetic carbon dioxide (CO₂) uptake may, in part, offset respiratory losses. To determine the effects of air and soil warming on CO₂ exchange in tundra, we established an ecosystem warming experiment – the Carbon in Permafrost Experimental Heating Research (CiPEHR) project – in the northern foothills of the Alaska Range in Interior Alaska. We used snow fences coupled with spring snow removal to increase deep soil temperatures and thaw depth (winter warming) and open‐top chambers to increase growing season air temperatures (summer warming). Winter warming increased soil temperature (integrated 5–40 cm depth) by 1.5 °C, which resulted in a 10% increase in growing season thaw depth. Surprisingly, the additional 2 kg of thawed soil C m^?2 in the winter warming plots did not result in significant changes in cumulative growing season respiration, which may have been inhibited by soil saturation at the base of the active layer. In contrast to the limited effects on growing‐season C dynamics, winter warming caused drastic changes in winter respiration and altered the annual C balance of this ecosystem by doubling the net loss of CO₂ to the atmosphere. While most changes to the abiotic environment at CiPEHR were driven by winter warming, summer warming effects on plant and soil processes resulted in 20% increases in both gross primary productivity and growing season ecosystem respiration and significantly altered the age and sources of CO₂ respired from this ecosystem. These results demonstrate the vulnerability of organic C stored in near surface permafrost to increasing temperatures and the strong potential for warming tundra to serve as a positive feedback to global climate change.     

The use of open-top chambers in forests for evaluating warming effects on herbaceous understorey plants

Open-top chambers (OTCs) are widely used experimental warming devices in open-field ecosystems such as tundra and alpine heath. However, knowledge of their performance in temperate deciduous forest ecosystems is largely lacking. The application of OTCs in forests might become important in the future since the effects of climate warming on growth, reproduction, and future distribution of understorey forest herbs have rarely been investigated. Therefore, polycarbonate OTCs covered with (OTCs+GF) and without permeable polypropylene GardenFleece (OTCs−GF) were installed in a temperate deciduous forest to create an experimental warming gradient. Short-term responses in phenology, growth, and reproduction of a model understorey forest herb (Anemone nemorosa L.) to OTC installation were determined. In a second growing season, an in-depth study of multiple abiotic conditions inside OTCs−GF was performed. Both OTCs+GF and OTCs−GF raised air and soil temperature in a realistic manner (ca. +0.4°C to +1.15°C), but OTCs−GF only in the leafless period (up to +1.5°C monthly average soil temperature). The early flowering forest herb A. nemorosa also showed a clear phenotypic response to OTC installation. Based on these facts and the large ecological drawbacks associated with OTCs+GF (mostly in connection with a higher relative air humidity and a lower light quantity) and very modest abiotic changes in OTCs−GF, we encourage the use of OTCs−GF in deciduous forest ecosystems for evaluating climate-warming effects on early flowering understorey forest herbs. There is also a potential to use this warming method on later flowering species, but this needs further research.     

Extreme rainfall events and cooling of sea turtle clutches: Implications in the face of climate warming

Understanding how climate change impacts species and ecosystems is integral to conservation. When studying impacts of climate change, warming temperatures are a research focus, with much less attention given to extreme weather events and their impacts. Here, we show how localized, extreme rainfall events can have a major impact on a species that is endangered in many parts of its range. We report incubation temperatures from the world's largest green sea turtle rookery, during a breeding season when two extreme rainfall events occurred. Rainfall caused nest temperatures to drop suddenly and the maximum drop in temperature for each rain‐induced cooling averaged 3.6°C (n = 79 nests, min = 1.0°C, max = 7.4°C). Since green sea turtles have temperature‐dependent sex determination, with low incubation temperatures producing males, such major rainfall events may have a masculinization effect on primary sex ratios. Therefore, in some cases, extreme rainfall events may provide a “get‐out‐of‐jail‐free card” to avoid complete feminization of turtle populations as climate warming continues.     

Deeper Snow Enhances Winter Respiration from Both Plant-associated and Bulk Soil Carbon Pools in Birch Hummock Tundra

It has only recently become apparent that biological activity during winter in seasonally snow-covered ecosystems may exert a significant influence on biogeochemical cycling and ecosystem function. One-seventh of the global soil carbon pool is stored in the bulk soil component of arctic ecosystems. Consistent climate change predictions of substantial increases in winter air temperatures and snow depths for the Arctic indicate that this region may become a significant net annual source of CO₂ to the atmosphere if its bulk soil carbon is decomposed. We used snow fences to investigate the influence of a moderate increase in snow depth from approximately 0.3 m (ambient) to approximately 1 m on winter carbon dioxide fluxes from mesic birch hummock tundra in northern Canada. We differentiated fluxes derived from the bulk soil and plant-associated carbon pools using an experimental ‘weeding’ manipulation. Increased snow depth enhanced the wintertime carbon flux from both pools, strongly suggesting that respiration from each was sensitive to warmer soil temperatures. Furthermore, deepened snow resulted in cooler and relatively stable soil temperatures during the spring-thaw period, as well as delayed and fewer freeze–thaw cycles. The snow fence treatment increased mean total winter efflux from 27 to 43 g CO₂-C m⁻². Because total 2004 growing season net ecosystem exchange for this site is estimated at 29–37 g CO₂-C m⁻², our results strongly suggest that a moderate increase in snow depth can enhance winter respiration sufficiently to switch the ecosystem annual net carbon exchange from a sink to source, resulting in net CO₂ release to the atmosphere.     

The limit to the distribution of a rainforest marsupial folivore is consistent with the thermal intolerance hypothesis

Models of impacts of climate change on species are generally based on correlations between current distributions and climatic variables, rather than a detailed understanding of the mechanisms that actually limit distribution. Many of the vertebrates endemic to rainforests of northeastern Australia are restricted to upland forests and considered to be threatened by climate change. However, for most of these species, the factors controlling their distributions are unknown. We examined the role of thermal intolerance as a possible mechanism limiting the distribution of Pseudochirops archeri (green ringtail possum), a specialist arboreal folivore restricted to rainforests above an altitude of 300 m in Australia’s Wet Tropics. We measured short-term metabolic responses to a range of ambient temperatures, and found that P. archeri stores heat when ambient temperatures exceed 30°C, reducing water requirements for evaporative cooling. Due to the rate at which body temperature increases with ambient temperatures >30°C, this strategy is not effective over periods longer than 5 h. We hypothesise that the distribution of P. archeri is limited by interactions between (i) the duration and severity of extreme ambient temperatures (over 30°C), (ii) the scarcity of free water in the rainforest canopy in the dry season, and (iii) constraints on water intake from foliage imposed by plant secondary metabolites and fibre. We predict that dehydration becomes limiting for P. archeri where extreme ambient temperatures (>30°C) persist for more than 5 h per day over 4–6 days or more. Consistent with our hypothesis, the abundance of P. archeri in the field is correlated with the occurrence of extreme temperatures, declining markedly at sites where the average maximum temperature of the warmest week of the year is above 30°C. Assuming the mechanism of limitation is based on extreme temperatures, we expect impacts of climate change on P. archeri to occur in discrete, rapid events rather than as a slow contraction in range.     

Responses to ozone in 12 provenances of European beech (Fagus sylvatica): genotypic variation and chamber effects on photosynthesis and dry-matter partitioning

Seedlings of 12 provenances of European beech ( Fagus sylvatica ) were exposed to ambient, non-filtered air (NF) or NF+50 nl l⁻¹ ozone (NF50) for 8 h d⁻¹ in open-top chambers (OTCs), from 1 June to 4 October 1995. In 1996 exposure was continued from 31 May to 1 October at four levels: charcoal-filtered air (CF), NF, NF50 and NF+100 nl l⁻¹ ozone (NF100). Provenances were grown for both seasons in outside reference plots. All treatments were replicated. Ozone did not affect gas exchange in the provenances until late in the second season. NF100 reduced photosynthesis by 18% in August 1996 compared to CF. In September, photosynthesis was reduced by 22% in NF50 and by 29% in NF100. After two seasons, ozone reduced the root:shoot ratio by 24% when comparing CF and NF100; this was caused by reductions in the root biomass. Ozone did not affect height growth or stem diameter, and there were no ozone×provenance interactions for any growth parameter. There was, however, a significant ozone×provenance interaction for photosynthesis, showing northwest European provenances to be more sensitive to ozone than southeast European provenances when comparing dose–response estimates. This is interpreted in terms of genetic adaptation of the photosynthetic apparatus to regional growing conditions. Seedlings in the chambers grew 45% taller, and had 28% more shoot biomass and 29% smaller root biomass, resulting in a 44% reduction of root:shoot ratios compared to seedlings outside. Increased temperature and decreased PAR inside the chambers relative to the outside were probably the main causes for the differences. The magnitude of the chamber effects in OTCs raises doubts about conclusions drawn from ozone exposures in such chambers. This and previous ozone experiments with OTCs may have reached inaccurate conclusions concerning the size of ozone responses due to chamber effects.     

Influence of common preanalytical variations on the metabolic profile of serum samples in biobanks

A blood pre-centrifugation delay of 24 h at room temperature influenced the proton NMR spectroscopic profiles of human serum. A blood pre-centrifugation delay of 24 h at 4°C did not influence the spectroscopic profile as compared with 4 h delays at either room temperature or 4°C. Five or ten serum freeze–thaw cycles also influenced the proton NMR spectroscopic profiles. Certain common in vitro preanalytical variations occurring in biobanks may impact the metabolic profile of human serum.     

Leaf temperatures in glasshouses and open-top chambers

Climate manipulation experiments are of key importance in identifying possible responses of plant communities and ecosystems to climate change. Experiments for warming the air under sunlit conditions are carried out in (partial) enclosures. These inevitably alter the energy balance inside, potentially altering tissue temperatures which affect metabolism and growth. Using an empirically validated energy balance model, we investigate effects of two widely used warming methods, climate-controlled glasshouses and passively warmed open-top chambers (OTCs), on leaf temperatures. The model applies standard energy balance formulas, supplemented with data on optical properties of glasshouse materials and wind conditions inside OTCs. Results show that the different radiation environment inside glasshouses did not produce large leaf temperature deviations compared with outside. Poor glasshouse design with significant radiation blockage by the structure or with insufficient ventilation did affect tissue temperatures more significantly. The drastic wind speed reduction inside OTCs approximately doubled the actual (canopy) warming compared with earlier reported increases in air temperature provided by this technique - an effect that was inflated if the plants' stomates closed. These results demonstrate that leaf temperatures were higher than previously considered in OTCs but not in climate-controlled glasshouses.     

Multi-objective environment chamber system for studying plant responses to climate change

This paper describes the technical information and performance of a new multi-objective chamber system enabling the control of environmental variables (e.g., temperature, CO₂, air humidity, wind speed, and UV-B radiation) for understanding plant responses to climate change. Over a whole growing season, four different climate scenarios were evenly programmed into the system’s 16 chambers as ambient environment (AMB), elevated temperature (ET), elevated CO₂ concentration (EC) and elevated temperature and CO₂ concentration (ETC). Simultaneously, the chamber effects were assessed regarding the physiological responses and growth of a boreal perennial grass (reed canary grass, Phalaris arundinacea L.). During the growing season, the chamber system provided a wide variety of climatic conditions for air temperature (T _a), relative humidity (RH) and CO₂ concentration (C _a) in the AMB chambers following outside conditions. The target temperature (+3.5°C) was achieved to a good degree in the ET and ETC chambers, being on average 3.3°C and 3.7°C higher than ambient conditions, respectively. The target concentration of CO₂ (700 ppm) was also well achieved in the EC and ETC chambers, being on average 704 ppm and 703 ppm, respectively. The stable airflow condition inside all of the chambers provided a homogeneous distribution of gases and temperature. The decreases in RH and increases in vapour pressure deficit (VPD) in the elevated temperature chambers were also maintained at a low level. Chamber effects were observed, with some physiological and growth parameters of plants being significantly lower in the AMB chambers, compared to outside conditions. The plant growth was negatively affected by the reduced radiation inside the chambers.     

Effects of temperature on performance and phenotypic selection on plant traits in alpine Ranunculus acris

Discovering temperature effects on the performance of tundra plants is important in the light of expected climate change. In this 4-year study on alpine Ranunculus acris, I test the hypothesis that temperature influences flowering phenology, reproductive success, growth, population dynamics, and phenotypic selection on quantitative traits, by experimental warming using open-top chambers (OTCs). Warming significantly advanced flowering phenology in only one season. Seed number and weight were significantly increased by warming during the first three seasons, but not in the fourth. Plants inside OTCs produced bigger leaves than control plants in the fourth season, but leaf number was unaffected by the OTC treatment. Despite increased seed number and weight, the density of flowering plants decreased inside OTCs compared to control plots, possibly because of a higher graminoid cover inside OTCs. Phenotypic-selection regression showed a significant selection differential and gradient in the direction of larger leaf sizes in control plants, whereas no selection on leaf size was detected on warmed plants. The direction and strength of selection on flowering time, flower number, and leaf number did not differ between control and warmed plants. The results suggest that increased reproductive output of R. acris may not be sufficient to maintain current population density under a denser vegetation cover. Received: 1 December 1998 / Accepted: 14 April 1999     

Responses of white spruce (Picea glauca) to experimental warming at a subarctic alpine treeline

From 2001 to 2004 we experimentally warmed 40 large, naturally established, white spruce [Picea glauca (Moench) Voss] seedlings at alpine treeline in southwest Yukon, Canada, using passive open‐top chambers (OTCs) distributed equally between opposing north and south‐facing slopes. Our goal was to test the hypothesis that an increase in temperature consistent with global climate warming would elicit a positive growth response. OTCs increased growing season air temperatures by 1.8°C and annual growing degree‐days by one‐third. In response, warmed seedlings grew significantly taller and had higher photosynthetic rates compared with control seedlings. On the south aspect, soil temperatures averaged 1.0°C warmer and the snow‐free period was nearly 1 month longer. These seedlings grew longer branches and wider annual rings than seedlings on the north aspect, but had reduced Photosystem‐II efficiency and experienced higher winter needle mortality. The presence of OTCs tended to reduce winter dieback over the course of the experiment. These results indicate that climate warming will enhance vertical growth rates of young conifers, with implications for future changes to the structure and elevation of treeline contingent upon exposure‐related differences. Our results suggest that the growth of seedlings on north‐facing slopes is limited by low soil temperature in the presence of permafrost, while growth on south‐facing slopes appears limited by winter desiccation and cold‐induced photoinhibition.     

Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.     

Stability Assessment of Injectable Castor Oil-Based Nano-sized Emulsion Containing Cationic Droplets Stabilized by Poloxamer–Chitosan Emulsifier Films

The objectives of the present work were to prepare castor oil-based nano-sized emulsion containing cationic droplets stabilized by poloxamer–chitosan emulgator film and to assess the kinetic stability of the prepared cationic emulsion after subjecting it to thermal processing and freeze–thaw cycling. Presence of cryoprotectants (5%, w/w, sucrose +5%, w/w, sorbitol) improved the stability of emulsions to droplet aggregation during freeze–thaw cycling. After storing the emulsion at 4°C, 25°C, and 37°C over a period of up to 6 months, no significant change was noted in mean diameter of the dispersed oil droplets. However, the emulsion stored at the highest temperature did show a progressive decrease in the pH and zeta potential values, whereas the emulsion kept at the lowest temperatures did not. This indicates that at 37°C, free fatty acids were formed from the castor oil, and consequently, the liberated free fatty acids were responsible for the reduction in the emulsion pH and zeta potential values. Thus, the injectable castor oil-based nano-sized emulsion could be useful for incorporating various active pharmaceutical ingredients that are in size from small molecular drugs to large macromolecules such as oligonucleotides.