A new quantitative method to measure activity of ice structuring proteins using differential scanning calorimetry

There are very few quantitative assays to measure the activity of antifreeze proteins (AFPs, or Ice Structuring Proteins, ISPs) and these can be prone to various inaccuracies and inconsistencies. Some methods rely only on unassisted visual assessment. When microscopy is used to measure ice crystal size, it is critical that standardized procedures be adopted, especially when image analysis software is used to quantify sizes. Differential Scanning Calorimetry (DSC) has been used to measure the thermal hysteresis activity (TH) of AFPs. In this study, DSC was used isothermally to measure enthalpic changes associated with structural rearrangements as a function of time. Differences in slopes of isothermal heat flow vs. time between winter wheat ISP or AFP type I containing samples, and those without ISP or AFP type I were demonstrated. ISP or AFP type I containing samples had significantly higher slopes compared to those without ISP or AFP type I. Samples with higher concentration of ISP or AFP type I showed higher slope values during the first hour and took up to 3 hr to attain equilibrium. Differences were attributed to activity of the proteins at the ice interface. Proteinaceous activity of ISPs or AFP type I was confirmed by loss of activity after treatment with protease.     

Relationships between cold hardiness, and ice nucleating activity, glycerol and protein contents in the hemolymph of caterpillars, Aporia crataegi L

Insects in Siberia must tolerate some of the coldest conditions on earth. The relationship between hemolymph ice nucleating activity, glycerol and total protein concentrations, and cold hardiness was explored in Aporia crataegi L. (Lepidoptera: Pieridae). Cold-hardened overwintering caterpillars were collected at a time of year when temperatures are regularly below -50 degree C, and warm-acclimated at +22 degree C, to see how changes in the physical and chemical properties of the hemolymph influence their cold hardiness potential. Warm acclimation led to a decrease in glycerol and proteins content in the hemolymph, which was associated with the decrease in ice nucleating activity and dramatic loss of cold hardiness potential of the caterpillars. It is suggested that one of the effects of cryoprotection in the freeze tolerant insects, caused by glycerol, might be associated with its ability to form larger aggregates of ice nucleating polypeptides that initiate the ice nucleation at high subzero temperatures. Such ice nucleating structures seem to ensure a high probability of ice nucleation at relatively high temperatures, which may contribute to the extraordinary cold hardiness of A. crataegi caterpillars, which may tolerate temperatures below -85 degree С.     

The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifoliata (L.)

The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 g water per g dry mass is believed to increase cytoplasmic viscosity. Cooling to -196 degree C was done at rates averaging between 0.17 and 1300 degree C per second, and warming at 600 or 1.35 degree C per second. Survival was assessed after 4 weeks in vitro. Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70 percent normal development ranged between 0.17 and about 1300 degree C per second for axes at a water content of 0.26 g water per g dry mass narrowing with increasing hydration to an apparent optimum at about 686 degree C per second in axes at 0.8 g water per g dry mass At 1.7 g water per g dry mass, axes cooled at 0.17 degree C per second yielded nearly 40 percent normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.     

Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686

The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.     

The effect of antifreeze proteins and poly(vinyl alcohol) on the nucleation of ice: a preliminary study

Three substances have been tested for ice nucleation inhibition. These were an antifreeze protein AFP III from the fish Macrozoarces americanus, an antifreeze glycoprotein AFGP from the fish Dissostichus mawsoni, and an 80% hydrolysed poly(vinyl alcohol) with a molecular weight of 9 to 10 kD. A nucleation spectrometer was used to test nucleation inhibition at a range of concentrations against two types of ice nuclei: those present in tap water and a bacterial nucleator from Pseudomonas syringae. The PVA reduced the nucleation temperature of tap water and the bacterial dispersions at all the concentrations which were tested. The AFGP reduced the nucleation temperature of tap water but enhanced the nucleation activity of the bacterial nucleators. At low concentrations the AFP III reduced the nucleation temperature of both tap water and the bacterial nucleator. At high concentrations the AFP III enhanced the nucleation temperature of the bacterial nucleator and broadened the nucleation spectrum of the tap water to encompass the nucleation spread of the control. The possible mechanisms of nucleation suppression and enhancement are discussed.     

The peculiarities of water crystallization and ice melting processes in the roots of one-year plants (Plantago major L.)

Results are presented of a water phase transition study in plantain (Plantago major L.) roots, which were used as a model system to research the peculiarities of water crystallization and ice melting processes in complex heterogeneous biological systems. It was confirmed that water in such systems is crystallized in two clearly distinguished temperature ranges: -10 to -25 degree capital ES, Cyrillic and -25 to -45 degree capital ES, Cyrillic. These water fractions are conditionally attributed to extracellular (-10 to -25 degree capital ES, Cyrillic) and intracellular (-25 to -45 degree capital ES, Cyrillic) solutions. A possible explanation is given for such significant supercooling of the intracellular solution. The values of osmotic pressures of extra- and intracellular solutions were determined according to ice melting curves. It is noted that the intracellular solution, which crystallized at lower temperatures, had a lower osmotic pressure.     

Cryoprotective effect of an insect antifreeze protein MpAFP 698 and its mutants from the desert beetle Microdera punctipennis

An insect antifreeze protein, MpAFP698, from the desert beetle Microdera punctipennis, shares 77% similarity with TmTHP (YL-3) from Tenebrio molitor. The predicted tertiary structure for MpAFP698 was modeled as a regular A-helix with TCT motifs arrayed to form the putative ice-binding surface. This model was validated via site-directed mutagenesis. The results suggest that the desert beetle in central Asia has evolved antifreeze proteins similar to those from North America continent. In an additional study, the recombinant MpAFP698 and its mutants were expressed in Escherichia coli strain BL21 (DE3). The thermal hysteresis activity of MpAFP698 was 1.73 degree C at 1.0 mg/ml. MpAFP698 was also tested by in vitro antifreeze activity assay to evaluate its cryoprotective effect at -20 degree C. MpAFP698 displays high cryoprotective effect on bacteria cells at freezing temperatures.     

Effect of intra- and extra-liposomal distribution of sodium chloride on the stability of large unilamellar vesicles

Three groups of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicle (LUV) dispersions were studied: LUV (A) dispersions with only extraliposomal sodium chloride (NaCl), LUV (B) dispersions with intra- and extraliposomal NaCl, and LUV (C) dispersions with only intraliposomal NaCl. The NaCl concentrations ranged from 0 to 150 mM. An abrupt increase in leakage was observed around -10 degree C for all the three groups of LUV, which coincided with the temperature of extraliposomal ice formation. Within the three groups, leakage of LUV (C) was significantly higher than the other groups. Extraliposomal ice formation and the resulting freeze-concentration of LUV may be the major cause of the leakage. Intraliposomal ice formation observed at -43 degree C seemed to stop leakage of LUV when LUV were frozen below -43 degree C. An exotherm of eutectic crystallization of NaCl was occasionally observed at -37 degree C, with a higher probability of formation at 150 mM extraliposomal NaCl than at 50 mM. The eutectic crystals were thought to cause additional leakage from the LUV (B).     

Purification and partial characterization of thermal hysteresis proteins from overwintering larvae of pine needle gall midge,Thecodiplosis japonensis (Diptera: cecidomiidae)

The pine needle gall midge of Thecodiplosis japonensis is a serious forest pest and overwinters as a 3rd instar larva at soil surface in Korea. The time necessary for killing 50% of larvae at -15 degree C is 160 min. During overwintering period, T. japonensis larvae accumulate relatively high content of trehalose as the main cryoprotectant. In this paper, the proteinaceous cryoprotectants were identified. Two thermal hysteresis proteins (THP-1S and 2S) were purified from overwintering larvae by ethanol fractionation, trichloroacetic acid precipitation, ion-exchange chromatography (DEAE-Sephadex A-25) and gel permeation chromatography (Sephadex G-100). Their molecular weights are 34.9 and 37.8 kD respectively. T. japonensis THPs cannot be stained by periodic acid-Schiffs' reagent, suggesting no carbohydrate in them. The thermal hysteresis activity of THP-2 at the concentration of 50 mg/ml is 11.02 +/- 0.08 degree C (mean +/- SD, n=10), perhaps the highest active insect THP. It is the first report of purified T. japonensis THPs in Diptera.     

Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida

The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.     

The use of calcination in exposing the entrapped Fe particles from multi-walled carbon nanotubes grown by chemical vapour deposition

Multi-walled carbon nanotubes (MWCNTs) were synthesized a by chemical vapour deposition method. The effect of calcination at temperatures ranging from 300 to 550°C in exposing the metal nanoparticles within the nanotube bundles was studied. The degree of degradation of the structural integrity of the MWCNTs during the thermal process was studied by Raman spectroscopy, X-ray diffraction analysis, field-emission scanning electron microscopy, and transmission electron microscopy. The thermal behaviour of the as-prepared and calcined samples was investigated by thermogravimetric analysis. Calcination in air, at 400°C for 1 h, was found to be an efficient and simple method to extract metallic impurities from the amorphous carbon shells with minimal damage to the tube walls and lengths. The nanotubes were observed to be damaged at temperatures higher than 450°C.     

Threonine side chain conformational population distribution of a type I antifreeze protein on interacting with ice surface studied via ¹³C-¹⁵N dynamic REDOR NMR

Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr ¹³C_γ and ¹⁵N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. χ-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.     

Design and performance analysis of smart photonic sensors for industrial applications

Toxic gas has a median fatal concentration in the oxygen of much more than 200 parts per million (ppm) but far less than 2000 ppm by volume of gas. Many industries, mines and thermal plants emit perilous gases that are more harmful to our human life. The Proposed nanosensor senses the various perilous gases and averts many accidents. In this paper, a two-dimensional Photonic Crystal (2D-PhC) resonator and PhC-based poisonous gas sensor based on the hexagonal and square crystal lattice are built-in smart way. The PhCs are artificial constructs of any material with an occasional enunciation of refractive index (RI). It has effective light manipulation and it would be helpful to obtain light migration in the handling of sensing applications. The TE/TM wave transmission can shift as per the RI value of different gases in the PhCs. The wavelength variations obtained agree well with the Finite Difference Time Domain (FDTD) study, and the simulation is performed by the tool RSoft. The spectral variables such as quality factor (QF), sensitivity (Se), transmitted output power and detection limit (DL) are evaluated using the RI value over the spectrum of different toxic gases. The proposed square crystal structure have acquired a QF range of 500.6, high efficiency of 99%, and a better Se of 716.6 nm/RIU at 1502 nm. The designed hexagonal crystal structure have acquired a QF range of 165.8, high efficiency of 99%, and a better Se of 798.24 nm/RIU at 1630 nm respectively. The DL for both the proposed sensors is very low. So, the designed smart sensor helps promptly recognize the contaminated gases in several places. The proposed nanosensor is helpful in industrial safety, health care applications, aerospace, agricultural, transportation, environmental monitoring, thermal plants and mines.     

Failure of thin films: Optical shearography versus electrochemical impedance spectroscopy

In the present work, the temperature versus thermal deformation (strain) with respect to time, of different coating films, was studied by a non-destructive technique (NDT) known as shearography. An organic coating, i.e., ACE Premium Enamel, on a metallic alloy, i.e., a carbon steel, was investigated at a temperature range simulating the severe weather temperatures in Kuwait, especially between the daylight and the nighttime temperatures, 20-60 °C. The investigation focused on determining the in-plane displacement of the coating, which amounts to the thermal deformation (strain) with respect to the applied temperature range. Furthermore, the investigation focused on determining the thermal expansion coefficients of coatings, the slope of the plot of the thermal deformation (strain) versus the applied temperature range. In other words, one could determine, from the decreasing value of the thermal expansion coefficients of coatings, a critical (steady state) value of the thermal expansion coefficients of coatings, in which the integrity of the coatings can be assessed with respect to time. In fact, determination of the critical (steady state) value of the thermal expansion coefficients of coatings could be accomplished independent of parameters, i.e., ultraviolet (UV) exposure, humidity, and exposure to chemical species, which normally are considered in conventional methods of assessing the integrity of coatings. Furthermore, results of shearography indicate that the technique is a very useful NDT method not only to determine the critical value of the thermal expansion coefficients of different coatings but also to be used as a 2D-microscope for monitoring the deformation of the coatings in real time at a submicroscopic scale. Also, the obtained data of the shearography technique were compared with data obtained by electrochemical impedance spectroscopy (EIS) in an aqueous solution of 3% NaCl.     

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.     

球等鞭金藻细胞生长抑制物的分离纯化

以球等鞭金藻老化培养液的无细胞上清处理液为研究对象,对存在于球等鞭金藻老化培养液中的抑制细胞生长的活性物质进行了分离纯化与抑制活性研究。运用乙酸乙酯萃取,紫外光谱分析,SephadexG-15凝胶过滤,制备薄层层析和C₁₈反相高效液相等光谱分析与纯化方法对其进行活性追踪的分离与纯化。结果表明,采用乙酸乙酯萃取球等鞭金藻老化培养液的无细胞上清处理液可以获得具有明显细胞抑制活性的细胞生长抑制物的粗提物,经200～400 nm的紫外光谱扫描,确定了细胞生长抑制物的特征吸收波长为235 nm。将粗提物用0.2 mol·L-1 NaOH溶解,经蒸馏水适当稀释后,以5%床体积的量加载于SephadexG-15凝胶层析柱并用蒸馏水进行洗脱。在紫外235 nm下,粗提物经SephadexG-15凝胶过滤,获得3个具有细胞抑制活性的组分。用1 mol·L-1 HCl将具有细胞抑制活性的组分pH调至2～3之间,用乙酸乙酯提取。于40 ℃下减压浓缩,将样品点在硅胶G板上,以氯仿为展开剂,进行薄层层析分离与制备,获得4个组分。经检测,仅R_f值为0.63的组分具有明显的细胞抑制活性。此活性组分采用硅烷化键合相C₁₈反相色谱柱,乙腈-水(体积比为63∶37)流动相,0.3 mL·min-1恒流洗脱,检测波长UV-235 nm,获得较好的分离效果。     

Pressure-induced transformation plasticity of H(2)O ice

Transformation plasticity is a deformation mechanism which occurs during phase transformation of an externally stressed material. Pressure-induced transformation plasticity of ice could be relevant to the geology of the moons of the outer solar system, and has long been postulated to occur in olivine in the earth's interior. In the present work, ice specimens were subjected to isostatic pressure cycling between 0 and 300 MPa to induce reversible polymorphic transformation between ice I and II at 230 K. When a small uniaxial compressive stress was applied during cycling, the specimens exhibited a compressive strain (as large as 18.5% after a single cycle) proportional to the applied stress, in agreement with observations and theory for transformation plasticity of metals induced by polymorphic thermal cycling.     

Study of Phase Change and Supercooling in Micro-Channels by Infrared Thermography

The present work presents a fast and simple new experimental method, designed to enhance the observation and characterization of thermal phenomena at microscale. Supercooling of water was carried out in micro-channels and recorded with a high-frequency infrared camera. The method is based on the coupling of microfluidics, infrared thermography, and inverse techniques. The objective is to extract a maximum of information from the experiment to perform advanced characterization of the system. First, a thermal modeling of such a system was written, then the image processing from infrared recordings allowed estimating the thermal properties (diffusivity) and the source term (energy released by the phase change). The novelty of the approach is the ability of measuring the heat released by the phase change and using this displacement to calculate the ice front propagation velocity and thermal properties. This method is appropriate for many other applications and is mainly devoted to the characterization of fluids during phase change at microscale.     

Phase Behavior of Rapidly Quenched PVDF/PMMA Blends as Characterized by Raman Spectroscopy,X‐Ray Diffraction and Thermal Techniques

Although PVDF/PMMA blends have been studied extensively, the phase behavior as a function of melt quenching conditions has not been examined in detail in the past. In this paper we report our results on the isotropic blends of PVDF/PMMA quenched into ice water as well as on a casting roll set at 30°C in all composition ranges. The results confirm the miscibility of this blend for all composition ranges, although at high PVDF (～85%) concentration micro heterogeneities were evidenced through thermal analysis. Though pure PVDF is observed to be mostly in the α crystalline form, the addition of PMMA favors the β crystal structure in composition range 85/15–60/40. Ice water quenching yields amorphous blends containing more than 40% PMMA and these films are deemed good candidates for rubbery state processes (between T _g and T _cc), including tenter frame biaxial stretching, where they can be oriented significantly at these low temperatures while undergoing strain induced crystallization.     

时间域混合像元分析在海冰密集度变化研究中的应用

传统意义混合像元分析方法是对有不同光谱特征的地物进行分解(spectral mixture analysis,SMA),得到各个组分在该像元内所占的百分数。而将光谱域的混合像元分析延伸至时间域内(temporalmixture analysis,TMA),提取的表征时间特性的端元用于像元分解所得残差小于8.5%,说明TMA提取南极地区海冰密集度时间变化特性具有可行性。将获取的多年平均端元用于2005年和2010年海冰密集度数据,得到残差分别为(1.4±2.42)%和(1.7±2.87)%,高于多年平均残差精度(1±1.53)%,在一定程度上反映某年海冰密集度数据相对多年平均值的变化。因此,TMA为全球变暖背景下的海冰密集度的时空特性研究提供了新思路。