Supercooling,ice nucleation and crystal growth: a systematic study in plant samples

This paper presents an innovative technological platform which is based on infrared video recording and is used for monitoring multiple ice nucleation events and their interactions, as they happen in 96 well microplates. Thousands of freezing curves were obtained during this study and the following freezing parameters were measured: cooling rate, nucleation point, freezing point, solidus point, degree of supercooling, duration of dendritic phase and duration of crystal growth. We demonstrate the use of this platform in the detection of ice nuclei in plant samples. Future applications of this platform may include breeding for frost tolerance, cryopreservation, frozen food technology and atmospheric sciences.     

Late Holocene climate change and human impact recorded in a south Swedish ombrotrophic peat bog

A peat core from a raised bog in southern Sweden was examined (plant macrofossils, pollen/non-pollen microfossils, colorimetric humification, carbon/nitrogen ratios, bulk densities, loss on ignition) to investigate the effects of climate change and human impact on the plant species composition and carbon accumulation of the peat forming vegetation. ¹⁴C wiggle-match dating was applied for fine-resolution dating. Cooling at the start of the Little Ice Age was reflected by a decline of thermophilous trees from the pollen record between ca. cal AD 1275–1590, coinciding with increases in atmospheric Δ¹⁴C pointing to decreases of solar activity (Wolf and Spörer minimum). Human impact also decreased. Later, the effect of human impact was clearly visible in the form of a black brown, decomposed peat layer, the top of which marks a gap of more than 300 years. Artificial drainage probably caused secondary decomposition and oxidation of the peat. From ca. cal AD 1960 the water table of the peat bog was restored following construction of a road and raised bog vegetation regenerated.     

Extremotrophs, extremophiles and broadband pigmentation strategies in a high arctic ice shelf ecosystem

Remnant ice shelves along the northern coast of Ellesmere Island, Nunavut, Canada ( approximately 83 degrees N) provide a habitat for cryo-tolerant microbial mat communities. Bioassays of bacterial and primary production were undertaken to quantify the short-term physiological response of the mats to changes in key variables that characterize this cryo-ecosystem (salinity, irradiance and temperature). The heterotrophic versus autotrophic community responses to these stressors differed markedly. The heterotrophic bacteria were extremophilic and specifically adapted to ambient conditions on the ice shelf, whereas the autotrophic community had broader tolerance ranges and optima outside the ambient range. This latter, extremotrophic response may be partly due to a diverse suite of pigments including oligosaccharide mycosporine-like amino acids, scytonemins, carotenoids, phycobiliproteins and chlorophylls that absorb from the near UV-B to red wavelengths. These pigments provide a comprehensive broadband strategy for coping with the multiple stressors of high irradiance, variable salinity and low temperatures in this extreme cryo-environment.     

四种小鼠便秘模型建立方法的比较

目的i初步探讨四种小鼠便秘模型建模方法的效果。方法：40只小鼠随机分为正常对照组（8只）和实验组（32只）,实验组分别采用经口灌胃止泻剂复方地芬诺酯、禁水不禁食、经口灌胃胃粘膜保护剂硫糖铝及经口灌胃冰水四种方法建立小鼠便秘模型,处理后观察四种不同方法建立的小鼠便秘模型首粒黑便排便时间、2小时内排便粒数及排便重量的差异。结果：与正常对照组比较,灌胃复方地芬诺酯、禁水不禁食、硫糖铝及冰水法四种造模方法均可使小鼠首粒黑便排便时间显著延长、2小时内排便粒数及排便重量显著减少（P〈0．05）。但采用复方地芬诺酯、禁水不禁食法处理的小鼠较经口灌胃硫糖铝及经口灌胃冰水诱导的小鼠首粒黑便排便时间显著延长、2小时内排便粒数及排便重量亦显著减少（P〈0．01）。结论：采用复方地芬诺酯、禁水不禁食方法建立小鼠便秘模型更加简单、有效。     

Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8 min. For an initial population of 9.3 log CFU mL^?1, the cultivability was lost starting at 14 min and a loss of viability was observed after 16 min of ozone treatment at 0.45 mg L^?1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8 min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4 °C of dead cells treated for 16 min.     

Bovine Oocytes Cryoinjury and How to Improve Their Development Following Cryopreservation

Bovine oocytes are less likely to undergo successful cryopreservation than cleavage-stage embryos. Bovine oocytes characteristically contain high levels of lipids that represent one of the major obstacles limiting efficient cryopreservation. These droplets together with structures such as cumulus cells, zona pellucida, cytoplasm membrane, cortical granules, mitochondria, spindle, and cytoskeleton (microtubles and microfilaments) often incur serious damage during cooling and warming. The cryoinjury could, to some extent, be decreased by selection of proper permeable and non-permeable cryoprotectants, and of vitrification with high cooling and warming rates. Additionally, such measures may also enhance their cryotolerance as partial removal of cumulus cells, modification of oocyte membrane constituents, polarization of the cytoplasmic lipid droplets by centrifugation, and addition of cytoskeleton relaxants or ice blockers into vitrification solutions. The improvement in cryopreservation methodology for bovine oocytes will no doubt augment other technologies such as bovine cloning and the establishment of gene bank for transgenic cattle.     

Physical, chemical and microbiological quality of ice used to cool drinks and foods in Greece and its public health implications

Ice used for direct human consumption or to preserve foods and cool down drinks can be contaminated with pathogenic microorganisms and may potentially become a vehicle for consumer’s infection. To evaluate physical, chemical and microbiological quality of commercial ice and ice used for fish and seafood, 100 ice samples collected at 10 different retail points in the region of Epirus were studied. The following microbiological parameters were determined: Total coliforms, fecal coliforms, Salmonella spp., Shigella spp., Yersinia spp., Escherichia coli, Campylobacter sp., Vibrio cholerae, Aeromonas spp., Pseudomonas aeruginosa and Clostridium perfringens.E. coli was detected in 22% and coliforms were detected in 31% of samples. Samples in which coliforms were detected fail to meet the microbiological criteria specified by the drinking water legislation.Aeromonas spp., Shigella spp., Campylobacter sp. and V. cholerae were not detected. Spore forms of C. perfringens were prevalent at 35% and the psychotropic bacterium’s P. aeruginosa and Yersinia spp. were found only at three samples each.The presence of large numbers of coliforms as well as of other pathogenic strains suggested that commercial ice and ice used to make cool drinks or in preservation of fish and seafood may represent a potential hazard to the consumer. In view of the results reported herein, it is highly recommended that national regulatory guidelines should be established for the production of ice as long as regular inspections.     

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish¹, plants^{2, 3}, arthropods^{4, 5}, fungi⁶, and bacteria⁷. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested^8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears¹¹, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments.We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously^{9, 10}, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)¹². This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies'' group (Queens University)¹⁰. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs^{5, 13}. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations^{14, 15}. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber¹³. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere¹⁶.

The Cold Stage System

The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).     

Porcine small intestinal submucosa sheets as a scaffold for human bone marrow stem cells

Native small intestinal submucosa (SIS) sheet was prepared by removal of inside and outside layer of porcine jejunum. The acid treated SIS sheet was also prepared by dipping of native SIS sheet in acetic acid solution. The native or acid treated SIS sheets exhibited elastic and soft property on touch. The surface of native SIS sheet appears to be covered with thin and long collagen fibers entangled into networks. The fibers and fibrils at acid treated SIS sheet disappeared due to the acidic erosion of collagen fiber. The water uptake of acid treated SIS sheet (1300%) was higher than that of the native SIS sheet (500%). The cell morphology and proliferation of human bone marrow stem cells (hBMSCs) on SIS sheet was examined. The hBMSCs on the SIS sheet showed a flattened morphology, while cells in the polyglycolic acid (PGA) mesh showed rounded cell morphology. The cell viability on native or acid treated SIS sheet was higher than that of PGA mesh. The hBMSCs in both native and acid treated SIS sheet were grown at a similar rate. The number of adhering hBMSCs increased with incubation time. Thus, we could confirm that native or acid treated SIS sheet could act as a potential scaffold to enhance the hBMSCs proliferation by providing probably natural environments.     

Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars

A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n=10) from a total of 10 stud boars classified as "good"(n=5) or sub-standard (e.g., "bad" freezers, n=5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p>0.05). However, we identified significant differences (p<0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences.