Practical Considerations for Determination of Glass Transition Temperature of a Maximally Freeze Concentrated Solution

Glass transition temperature is a unique thermal characteristic of amorphous systems and is associated with changes in physical properties such as heat capacity, viscosity, electrical resistance, and molecular mobility. Glass transition temperature for amorphous solids is referred as (T _g), whereas for maximally freeze concentrated solution, the notation is (T _g′). This article is focused on the factors affecting determination of T _g′ for application to lyophilization process design and frozen storage stability. Also, this review provides a perspective on use of various types of solutes in protein formulation and their effect on T _g′. Although various analytical techniques are used for determination of T _g′ based on the changes in physical properties associated with glass transition, the differential scanning calorimetry (DSC) is the most commonly used technique. In this article, an overview of DSC technique is provided along with brief discussion on the alternate analytical techniques for T _g′ determination. Additionally, challenges associated with T _g′ determination, using DSC for protein formulations, are discussed. The purpose of this review is to provide a practical industry perspective on determination of T _g′ for protein formulations as it relates to design and development of lyophilization process and/or for frozen storage; however, a comprehensive review of glass transition temperature (T _g, T _g′), in general, is outside the scope of this work.     

Effect of Water Content on the Glass Transition Temperature of Calcium Maltobionate and its Application to the Characterization of Non-Arrhenius Viscosity Behavior

To understand the fundamental physical properties of calcium maltobionate (MBCa), its water sorption isotherm, glass transition temperature (T _g), and viscosity (η) were investigated and compared with those of maltobionic acid (MBH) and maltose. Although amorphous maltose crystalized at water activity (a _w) higher than 0.43, MBCa and MBH maintained an amorphous state over the whole a _w range. In addition, MBCa had a higher T _g and greater resistance to water plasticizing than MBH and maltose. These properties of MBCa likely originate from the strong interaction between MBCa and water induced by electrostatic interactions. Moreover, the effects of temperature and water content on η of an aqueous MBCa solution were evaluated, and its behavior was described using a semi-empirical approach based on a combination of T _g extrapolated by the Gordon-Taylor equation and a non-Arrhenius formula known as the Vogel–Fulcher–Tammann equation. This result will be useful for understating the effect of MBCa addition on the solution’s properties.     

Optimization of Component Yields and Thermal Properties by Organosolv Fractionation of Loblolly Pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) Using Response Surface Design

Lignin, a low value by-product of biomass fractionation, is of current particular interest for the production of value added materials such as carbon fibers within a biorefinery. Accordingly, we have isolated lignin, hemicellulose, and cellulose by organosolv fractionation of loblolly pine (Pinus taeda) under the influence different fractionation severities (2.50–3.31). A designed experimental matrix targeted maximum lignin and pulp yields and lignin glass transition temperature (T_g) as a function of several process parameters. Optimal fractionation conditions were estimated from an enhanced regression model of the statistically significant variables generated using response surface design (RSD). The most significant factor influencing lignin and pulp yields was solvent composition (p-value <?0.0001). A maximum lignin yield of 94.71 wt% and maximum cellulose yield of 74.96 wt% were determined at combined severities of 3.21 and 2.63, respectively. The lowest T_g of 132.83 °C was found at a combined severity of 2.82.     

Evolution of protein transport to the chloroplast envelope membranes

Comparing with other angiosperms, most members within the family Orchidaceae have lower photosynthetic capacities. However, the underlying mechanisms remain unclear. Cypripedium and Paphiopedilum are closely related phylogenetically in Orchidaceae, but their photosynthetic performances are different. We explored the roles of internal anatomy and diffusional conductance in determining photosynthesis in three Cypripedium and three Paphiopedilum species, and quantitatively analyzed their diffusional and biochemical limitations to photosynthesis. Paphiopedilum species showed lower light-saturated photosynthetic rate (A _N), stomatal conductance (g _s), and mesophyll conductance (g _m) than Cypripedium species. A _N was positively correlated with g _s and g _m. And yet, in both species A _N was more strongly limited by g _m than by biochemical factors or g _s. The greater g _s of Cypripedium was mainly affected by larger stomatal apparatus area and smaller pore depth, while the less g _m of Paphiopedilum was determined by the reduced surface area of mesophyll cells and chloroplasts exposed to intercellular airspace per unit of leaf area, and much thicker cell wall thickness. These results suggest that leaf anatomical structure is the key factor affecting g _m, which is largely responsible for the difference in photosynthetic capacity between those two genera. Our findings provide new insight into the photosynthetic physiology and functional diversification of orchids.     

Informative Value of Temperatures in Different Areas of the Human Body for Correcting Body Thermal Imbalance during Extravehicular Activities

Identification of noninvasive and informative sites on the body reflecting the development of body thermal imbalance during extravehicular activities (EVAs) is highly important for enhancing astronaut safety. Temperature changes were evaluated on several areas of the head (the mastoid fossa (T _mf ), the forehead (T _fo ), and the cheek (T _ch )) and on the fingers (T _fing ). Subjects were dressed in a multicompartment liquid cooling/warming garment. Studies I and II consisted of different combinations of hood versus garment cooling or warming imposed across stages; studies III and IV involved sagittally divided cooling or warming regimes with the hood worn (study III) or with the head uncovered (study IV). In studies I and II, T _mf significantly (P < 0.05) differed between stages 2, when the head was cooled and the rest of the body heated, and 3, when the head was heated and the rest of the body cooled. The T _mf changes were consistent with the thermal conditions imposed on the head but not reflective of the developing body heat deficit. In study III, the T _mf at stages 2 and 3 on the right or the left followed the thermal conditions on the ipsilateral side of the body (P < 0.01). In study IV, T _fing showed no significant differences across stages. In studies I–IV, T _fing showed consistent changes across stages (P < 0.05), reflecting the developing body heat deficit. In all studies, there were no significant differences in rectal temperature (T _re ) across stages. T _mf and temperatures at other head skin sites did not respond in accordance with the actual intensity of a heat or cold flux from the garment and were not reflective of the overall development of body thermal imbalance. T _fing was a more adequate indicator of initial thermal destabilization and provided information that would be useful for monitoring the thermal balance and comfort during EVAs.     

Preparation and Evaluation of Diosgenin Nanocrystals to Improve Oral Bioavailability

Diosgenin (DSG), a well-known steroid sapogenin derived from Dioscorea nipponica Makino and Dioscorea zingiberensis Wright, has a variety of bioactivities. However, it shows low oral bioavailability due to poor aqueous solubility and strong hydrophobicity. The present study aimed to develop DSG nanocrystals to increase the dissolution and then improve the oral bioavailability and biopharmaceutical properties of DSG. DSG nanocrystals were prepared by the media milling method using a combination of pluronic F127 and sodium dodecyl sulfate as surface stabilizers. The physicochemical properties of the optimal DSG nanocrystals were characterized using their particle size distribution, morphology, differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy data, and solubility and dissolution test results. Pharmacokinetic studies of the DSG coarse suspension and its nanocrystals were performed in rats. The particle size and polydispersity index of DSG nanocrystals were 229.0?±?3.7 nm and 0.163?±?0.064, respectively. DSG retained its original crystalline state during the manufacturing process, and its chemical structure was not compromised by the nanonizing process. The dissolution rate of the freeze-dried DSG nanocrystals was significantly improved in comparison with the original DSG. The pharmacokinetic studies showed that the AUC_0–72h and C _max of DSG nanocrystals increased markedly (p?<?0.01) in comparison with the DSG coarse suspension by about 2.55- and 2.01-fold, respectively. The use of optimized nanocrystals is a good and efficient strategy for oral administration of DSG due to the increased dissolution rate and oral bioavailability of DSG nanocrystals.     

Simple generalisation of a mesophyll resistance model for various intracellular arrangements of chloroplasts and mitochondria in C<Subscript>3</Subscript> leaves

The classical definition of mesophyll conductance (g _m) represents an apparent parameter (g _m,app) as it places (photo)respired CO₂ at the same compartment where the carboxylation by Rubisco takes place. Recently, Tholen and co-workers developed a framework, in which g _m better describes a physical diffusional parameter (g _m,dif). They partitioned mesophyll resistance (r _m,dif = 1/g _m,dif) into two components, cell wall and plasmalemma resistance (r _wp) and chloroplast resistance (r _ch), and showed that g _m,app is sensitive to the ratio of photorespiratory (F) and respiratory (R _d) CO₂ release to net CO₂ uptake (A): g _m,app = g _m,dif/[1?+?ω(F?+?R _d)/A], where ω is the fraction of r _ch in r _m,dif. We herein extend the framework further by considering various scenarios for the intracellular arrangement of chloroplasts and mitochondria. We show that the formula of Tholen et al. implies either that mitochondria, where (photo)respired CO₂ is released, locate between the plasmalemma and the chloroplast continuum or that CO₂ in the cytosol is completely mixed. However, the model of Tholen et al. is still valid if ω is replaced by ω(1?σ), where σ is the fraction of (photo)respired CO₂ that experiences r _ch (in addition to r _wp and stomatal resistance) if this CO₂ is to escape from being refixed. Therefore, responses of g _m,app to (F?+?R _d)/A lie somewhere between no sensitivity in the classical method (σ =1) and high sensitivity in the model of Tholen et al. (σ =0).     

Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

QbD-Oriented Development and Characterization of Effervescent Floating-Bioadhesive Tablets of Cefuroxime Axetil

The objective of the present studies was systematic development of floating-bioadhesive gastroretentive tablets of cefuroxime axetil employing rational blend of hydrophilic polymers for attaining controlled release drug delivery. As per the QbD-based approach, the patient-centric target product profile and quality attributes of tablet were earmarked, and preliminary studies were conducted for screening the suitability of type of polymers, polymer ratio, granulation technique, and granulation time for formulation of tablets. A face-centered cubic design (FCCD) was employed for optimization of the critical material attributes, i.e., concentration of release controlling polymers, PEO 303 and HPMC K100 LV CR, and evaluating in vitro buoyancy, drug release, and ex vivo mucoadhesion strength. The optimized formulation was embarked upon through numerical optimization, which yield excellent floatation characteristic with drug release control (i.e., T _60%?>?6 h) and bioadhesion strength. Drug-excipient compatibility studies through FTIR and P-XRD revealed the absence of any interaction between the drug and polymers. In vivo evaluation of the gastroretentive characteristics through X-ray imaging and in vivo pharmacokinetic studies in rabbits revealed significant extension in the rate of drug absorption (i.e., T _max, K _a, and MRT) from the optimized tablet formulation as compared to the marketed formulation. Successful establishment of various levels of in vitro/in vivo correlations (IVIVC) substantiated high degree of prognostic ability of in vitro dissolution conditions in predicting the in vivo performance. In a nutshell, the studies demonstrate successful development of the once-a-day gastroretentive formulations of cefuroxime axetil with controlled drug release profile and improved compliance.     

Analysis of applicability of triplet-state emission of molecular hydrogen for spectral diagnostics of a DC discharge

The applicability of emission of the N ³Λ_σ triplet states of molecular hydrogen for spectral diagnostics of the positive column of a dc glow discharge in hydrogen at translational gas temperatures of 360–600 K, specific absorbed powers of 0.8–4.25 W/cm, gas pressures of p = 0.3–15.0 Torr, reduced fields of E/N = 30–130 Td, and electron densities of n _e = 4.0 × 10⁹–6.5 × 10¹⁰ cm^–3 is analyzed by using an advanced level-based semi-empirical collisional?radiative model. It is found that secondary processes make the main contribution to the population and decay of the N ³Λ_σ = a ³Σ⁺ _g , c ³Π _u , g ³Σ⁺ _g , h ³Σ⁺ _g , and i ³Π _g triplet states. The dipole-allowed transitions e ³Σ⁺ _g → a ³Σ⁺ _g , f ³Σ⁺ _g → a ³Σ⁺ _g , g ³Σ⁺ _g and k ³Π _u → a ³Σ⁺ _g can be used for spectral diagnostics of a dc discharge within a simplified coronal model.     

Microwave electrode discharge in nitrogen: Structure and characteristics of the electrode region

The parameters of the electrode region of an electrode microwave discharge in nitrogen are studied by emission spectroscopy. The radial and axial distributions of the intensities of the bands of the second (N₂(C ³Π _u → B ³Π _g )) and first (N₂(B ³Π _g → A ³Σ _u ⁺ )) positive systems of molecular nitrogen and the first negative system of nitrogen ions (N ₂ ⁺ (B ²Σ _u ⁺ → X ²Σ _g ⁺ )), the radial profiles of the electric field E and the electron density N _e , and the absolute populations of the vibrational levels v _C = 0–4 of the C ³Π _u excited state of N₂ and the vibrational level v _Bi = 0 of the B ²Σ _u ⁺ excited state of a molecular nitrogen ion are determined. The population temperature of the first vibrational level T _V of the ground electronic state X ¹Σ _g ⁺ of N₂ and the excitation temperature T _C of the C ³Π _u state in the electrode region of the discharge are measured. The radius of the spherical region and the spatially integrated plasma emission spectra are studied as functions of the incident microwave power and gas pressure. A method for determining the electron density and the microwave field strength from the plasma emission characteristics is described in detail.     

Temperature sensitivity of soil respiration in a low-latitude forest ecosystem varies by season and habitat but is unaffected by experimental warming

Experimental warming of forest ecosystems typically stimulates soil respiration (CO₂ efflux), but most warming experiments have been conducted in northern latitudes (>?40°N) with relatively young soils. We quantified the influence of experimental warming on soil respiration (R_T) in two adjacent forest habitats—a mature, closed canopy forest and a gap where trees were manually removed— on highly-weathered Ultisols of the southeastern U.S. (33°N). Using temperature variation, both natural and induced by experimental warming, we also quantified the temperature sensitivity of R_T, defined as the activation energy, E_A in the Arrhenius equation. Experimental warming (either + 3 °C or + 5 °C above ambient) did not significantly increase soil respiration rate or cumulative CO₂ loss over the 3 years of the experiment, and did not influence the temperature sensitivity of soil respiration, once the influence of natural temperature variation was taken into consideration. Despite the absence of an experimental warming effect, we observed that E_A varied on monthly time scales, and varied differently in each habitat. Soil moisture and habitat also influenced R_T, but the effects were not consistent, and varied by month. Our results suggest that although R_T does depend on temperature, the sensitivity of R_T to temperature variation is influenced primarily by factors like microclimate and plant phenology that can change on relatively short (<?monthly) time scales. Thus, using the temperature sensitivity of R_T to predict future CO₂ losses due to warming is only reasonable if monthly variation in E_A is incorporated into models for lower-latitude subtropical ecosystems with highly weathered soils, such as those in this study. Finally, our results suggest that higher temperatures may not enhance R_T in highly-weathered, C-poor soils to the extent that has been reported in prior studies of high-latitude soils, which may constrain ecosystem-atmosphere carbon exchanges and feedbacks to the climate system.     

Hydrothermal time analysis of watermelon (<Emphasis Type="Italic">Citrullus vulgaris</Emphasis> cv. ‘Crimson sweet’) seed germination

This study evaluated the ability of a hydrothermal time model (HTT) to describe the kinetics of watermelon (Citrullus vulgaris cv. ‘Crimson sweet’) seed germination under different temperatures (T) and water potentials (ψ) and also to determine the cardinal temperatures of watermelon. Results indicated that ψ influenced germination rate and germination percentage. For this seed lot, cardinal temperatures were 10 °C for T _b, 28.34 °C for T _o and 40.8 °C for T _c in the control (0 MPa) treatment. There was a decrease in hydrotime constant (θ _H) when T was increased to T _o and then remained constant at supra-optimal temperatures (30 MPah^?1). Also, at temperatures above T _o, ψ _b(50) values increased linearly with T. The k _T value (the slope of the relationship between ψ _b(50) and T exceeds T _o) of this seed lot was calculated as 0.076 MPa°Ch^?1. Results this study show that when the HTT model is applied, it can accurately describe ψ _b(g) and the course of germination around Ts (R ² = 0.82). Moreover, the ψ _b(50) was estimated to be ?0.96 MPa based on this model. Consequently, the germination response of watermelon for all Ts and ψs can be adequately described by the HTT model and enabling it to be used as a predictive tool in watermelon seed germination simulation models.     

Mesophyll conductance to CO<Subscript>2</Subscript> in leaves of Siebold’s beech (<Emphasis Type="Italic">Fagus crenata</Emphasis>) seedlings under elevated ozone

Ozone is an air pollutant that negatively affects photosynthesis in woody plants. Previous studies suggested that ozone-induced reduction in photosynthetic rates is mainly attributable to a decrease of maximum carboxylation rate (V_cmax) and/or maximum electron transport rate (J_max) estimated from response of net photosynthetic rate (A) to intercellular CO₂ concentration (C_i) (A/C_i curve) assuming that mesophyll conductance for CO₂ diffusion (g_m) is infinite. Although it is known that C_i-based V_cmax and J_max are potentially influenced by g_m, its contribution to ozone responses in C_i-based V_cmax and J_max is still unclear. In the present study, therefore, we analysed photosynthetic processes including g_m in leaves of Siebold’s beech (Fagus crenata) seedlings grown under three levels of ozone (charcoal-filtered air or ozone at 1.0- or 1.5-times ambient concentration) for two growing seasons in 2016–2017. Leaf gas exchange and chlorophyll fluorescence were simultaneously measured in July and September of the second growing season. We determined the A, stomatal conductance to water vapor and g_m, and analysed A/C_i curve and A/C_c curve (C_c: chloroplast CO₂ concentration). We also determined the Rubisco and chlorophyll contents in leaves. In September, ozone significantly decreased C_i-based V_cmax. At the same time, ozone decreased g_m, whereas there was no significant effect of ozone on C_c-based V_cmax or the contents of Rubisco and chlorophyll in leaves. These results suggest that ozone-induced reduction in C_i-based V_cmax is a result of the decrease in g_m rather than in carboxylation capacity. The decrease in g_m by elevated ozone was offset by an increase in C_i, and C_c did not differ depending on ozone treatment. Since C_c-based V_cmax was also similar, A was not changed by elevated ozone. We conclude that g_m is an important factor for reduction in C_i-based V_cmax of Siebold’s beech under elevated ozone.     

Oral fast-dissolving films containing lutein nanocrystals for improved bioavailability: formulation development, <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> evaluation

Lutein is widely used as diet supplement for prevention of age-related macular degeneration. However, the application and efficacy of lutein in food and nutritional products has been hampered due to its poor solubility and low oral bioavailability. This study aimed to develop and evaluate the formulation of oral fast-dissolving film (OFDF) containing lutein nanocrystals for enhanced bioavailability and compliance. Lutein nanocrystals were prepared by anti-solvent precipitation method and then encapsulated into the films by solvent casting method. The formulation of OFDF was optimized by Box-Behnken Design (BBD) as follows: HPMC 2.05% (w/v), PEG 400 1.03% (w/v), Cremophor EL 0.43% (w/v). The obtained films exhibited uniform thickness of 35.64 ± 1.64 μm and drug content of 0.230 ± 0.003 mg/cm² and disintegrated rapidly in 29 ± 8 s. The nanocrystal-loaded films with reconstituted particle size of 377.9 nm showed better folding endurance and faster release rate in vitro than the conventional OFDFs with raw lutein. The microscope images, thermograms, and diffractograms indicated that lutein nanocrystals were highly dispersed into the films. After administrated to SD rats, t _max was decreased from 3 h for oral solution formulation to less than 0.8 h for OFDF formulations, and C _max increased from 150 ng/mL for solution to 350 ng/mL for conventional OFDF or 830 ng/mL for nanocrystal OFDF. The AUC _0-24h of conventional or nanocrystal OFDF was 1.37 or 2.08-fold higher than that of the oral solution, respectively. These results suggested that drug nanocrystal-loaded OFDF can be applied as a promising approach for enhanced bioavailability of poor soluble drugs like lutein.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Thermal Preference of the Bush Cricket <Emphasis Type="Italic">Isophya rizeensis</Emphasis>; Testing the Effect of Countergradient Selection

Thermal preference is one of the most crucial components of behavioral thermoregulation in ectotherms, and documenting the adaptation of thermal preference carries great importance for studying the evolution of thermal biology. However there are not many studies focusing on the adaptation of thermal preference in elevational and latitudinal gradients. Isophya rizeensis is a color polymorphic bush cricket species endemic to the mountainous region of northeastern Turkey. Populations of this species are distributed in a wide elevational range between 350 and 2300 m. In this study, we hypothesized that the thermal preference of Isophya rizeensis might follow a countergradient variation where crickets from higher altitudes have higher temperature preferences compared to crickets from lower altitudes. To test this hypothesis, thermal preference values (T _pref ) of crickets from three altitudes groups (low, middle and high) were measured with a thermal gradient experiment. Additionally, body temperatures (T _b ) and environmental temperatures (T _a ) were measured in field. Deviation values of T _b and T _a from T _pref were calculated to investigate the extent of thermoregulation. As Isophya rizeensis is color polymorphic species where morphology pattern changes from lighter to darker types with increasing altitude we also tested whether coloration has any effect on temperature excess (T _ex ) and thermoregulation. Thermal preference values did not differ significantly between three groups and also colouration does not influence the extent of thermoregulation in this species. These results indicate that there is not sufficient evidence for the existence of a countergradient selection related with thermal behavior. However, the deviation of body (D _b ) and environmental (D _a ) temperatures suggest that at higher altitudes thermoregulation might be more efficient than lower altitudes.     

Effects of endogenous salicylic acid synthesized through PAL and ICS pathway on baicalin and baicalein accumulation in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

The aim of this study was to investigate the effect of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS) on free salicylic acid (FSA) or total salicylic acid (TSA) content, and the effect of endogenous SA on baicalin and baicalein accumulation in Scutellaria baicalensis Georgi, respectively. We amplified partial sequences of PAL and ICS genes in Scutellaria baicalensis Georgi and silenced the two genes with virus-induced gene silence (VIGS) technique, respectively. The influence of gene silence on FSA, TSA, baicalin, and baicalein accumulation in Scutellaria baicalensis Georgi were analyzed, and these parameters were also investigated under high temperature. Results indicated that PAL silence significantly affected the FSA, ICS affected TSA content. FSA significantly affected the baicalin, rather than baicalein content. Our results along with previous studies indicated PAL and ICS were different in the regulation of FSA or TSA synthesis, and FSA and TSA were different in the regulation of baicalin and baicalein synthesis in Scutellaria baicalensis Georgi.     

The coupling effects of water deficit and nitrogen supply on photosynthesis,WUE, and stable isotope composition in <Emphasis Type="Italic">Picea asperata</Emphasis>

Water stress and nitrogen (N) availability are the two main factors limiting plant growth, and the two constrains can interact in intricate ways. Moreover, atmospheric N depositions are altering the availability of these limiting factors in many terrestrial ecosystems. Here, we studied the combined effects of different soil water availability and N supply on photosynthesis and water-use efficiency (WUE) in Picea asperata seedlings cultured in pots, using gas exchange, and stable carbon and nitrogen isotope composition (δ ¹³C and δ ¹⁵N). Photosynthesis under light saturation (A _sat) and stomatal conductance (g _s) of P. asperata decreased as the soil moisture gradually diminished. Under severe water-stress condition, N addition decreased the A _sat and g _s, whereas the positive effects were observed in moderate water-stress and well-watered conditions. The effect of N addition on the intrinsic WUE (WUE_i) deduced from gas exchange was associated with soil water availability, whereas long-term WUE evaluated by leaf δ ¹³C only affected by soil water availability, and it would be elevated with soil moisture gradually diminished. Water deficit would restrict the uptake and further transport of N to the aboveground parts of P. asperata, and then increasing δ ¹⁵N. Therefore, δ ¹⁵N in plant tissues may reflect changes in N allocation within plants. These results indicate that the effect of N enrichment on photosynthesis in P. asperata is largely, if not entirely, dependent on the severity of water stress, and P. asperata would be more sensitive to increasing N enrichment under low soil water availability than under high soil moisture.     

<Emphasis Type="Italic">Flavobacterium zaozhuangense</Emphasis> sp. nov., a new member of the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from metolachlor-contaminated soil

Strain ZZ-8^T, a Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented, rod-shaped bacterium, was isolated from metolachlor-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ZZ-8^T is a member of the genus Flavobacterium and shows high sequence similarity to Flavobacterium humicola UCM-46^T (97.2%) and Flavobacterium pedocola UCM-R36^T (97.1%), and lower (<?97%) sequence similarity to other known Flavobacterium species. Chemotaxonomic analysis revealed that strain ZZ-8^T possessed MK-6 as the major respiratory quinone; and iso-C_15:0 (28.5%), summed feature 9 (iso-C_17:1 w9c/C_16:0 10-methyl, 22.9%), iso-C_17:0 3-OH (17.0%), iso-C_15:0 3-OH (8.9%), iso-C_15:1 G (8.6%) and summed feature 3 (C_16:1 w7c/C_16:1 w6c, 5.7%) as the predominant fatty acids. The polar lipids of strain ZZ-8^T were determined to be lipids, a glycolipid, aminolipids and phosphatidylethanolamine. Strain ZZ-8^T showed low DNA–DNA relatedness with F. pedocola UCM-R36^T (43.23?±?4.1%) and F. humicola UCM-46^T (29.17?±?3.8%). The DNA G+C content was 43.3 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA–DNA hybridization, strain ZZ-8^T is considered a novel species of the genus Flavobacterium, for which the name Flavobacterium zaozhuangense sp. nov. (type strain ZZ-8^T?=?KCTC 62315 ^T?=?CCTCC AB 2017243^T) is proposed.