Temperature-sensitive paramagnetic liposomes for image-guided drug delivery: Mn versus [Gd(HPDO3A)(H2O)]

Temperature-sensitive liposomes (TSLs) loaded with doxorubicin (Dox), and Magnetic Resonance Imaging contrast agents (CAs), either manganese (Mn^2 +) or [Gd(HPDO3A)(H₂O)], provide the advantage of drug delivery under MR image guidance. Encapsulated MRI CAs have low longitudinal relaxivity (r₁) due to limited transmembrane water exchange. Upon triggered release at hyperthermic temperature, the r₁ will increase and hence, provides a means to monitor drug distribution in situ. Here, the effects of encapsulated CAs on the phospholipid bilayer and the resulting change in r₁ were investigated using MR titration studies and ¹H Nuclear Magnetic Relaxation Dispersion (NMRD) profiles. Our results show that Mn^2 + interacted with the phospholipid bilayer of TSLs and consequently, reduced doxorubicin retention capability at 37 °C within the interior of the liposomes over time. Despite that, Mn^2 +-phospholipid interaction resulted in higher r₁ increase, from 5.1 ± 1.3 mM^− 1 s^− 1 before heating to 32.2 ± 3 mM^− 1 s^− 1 after heating at 60 MHz and 37 °C as compared to TSL(Gd,Dox) where the longitudinal relaxivities before and after heating were 1.2 ± 0.3 mM^− 1 s^− 1 and 4.4 ± 0.3 mM^− 1 s^− 1, respectively. Upon heating, Dox was released from TSL(Mn,Dox) and complexation of Mn^2 + to Dox resulted in a similar Mn^2 + release profile. From 25 to 38 °C, r₁ of [Gd(HPDO3A)(H₂O)] gradually increased due to increase transmembrane water exchange, while no Dox release was observed. From 38 °C, the release of [Gd(HPDO3A)(H₂O)] and Dox was irreversible and the release profiles coincided. By understanding the non-covalent interactions between the MRI CAs and phospholipid bilayer, the properties of the paramagnetic TSLs can be tailored for MR guided drug delivery.     

Acid-activatable prodrug nanogels for efficient intracellular doxorubicin release

Endosomal pH-activatable doxorubicin (DOX) prodrug nanogels were designed, prepared, and investigated for triggered intracellular drug release in cancer cells. DOX prodrugs with drug grafting contents of 3.9, 5.7, and 11.7 wt % (denoted as prodrugs 1, 2, and 3, respectively) were conveniently obtained by sequential treatment of poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-co-ethyl glycinate methacrylamide) (PEG-b-P(HEMA-co-EGMA)) copolymers with hydrazine and doxorubicin hydrochloride. Notably, prodrugs 1, 2, and 3 formed monodispersed nanogels with average sizes of 114.4, 75.3, and 66.3 nm, respectively, in phosphate buffer (PB, 10 mM, pH 7.4). The in vitro release results showed that DOX was released rapidly and nearly quantitatively from DOX prodrug nanogels at endosomal pH and 37 °C in 48 h, whereas only a minor amount (ca. 20% or less) of drug was released at pH 7.4 under otherwise the same conditions. Confocal laser scanning microscope (CLSM) observations revealed that DOX prodrug nanogels delivered and released DOX into the cytosols as well as cell nuclei of RAW 264.7 cells following 24 h incubation. MTT assays demonstrated that prodrug 3 had pronounced cytotoxic effects to tumor cells following 72 h incubation with IC(50) data determined to be 2.0 and 3.4 μg DOX equiv/mL for RAW 264.7 and MCF-7 tumor cells, respectively. The corresponding polymer carrier, PEG-b-P(HEMA-co-GMA-hydrazide), was shown to be nontoxic up to a tested concentration of 1.32 mg/mL. These endosomal pH-activatable DOX prodrug nanogels uniquely combining features of water-soluble macromolecular prodrugs and nanogels offer a promising platform for targeted cancer therapy.     

In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes

Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with > 70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 °C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1 ± 4.0% of doxorubicin over 3 h at 37  °C in the presence of serum. The drug was almost completely released within 120s at 42 °C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 °C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.     

Enhanced resistance to peroxide stress in Escherichia coli grown outside their niche temperatures

Extended exposure of Escherichia coli to temperatures above and below their growth optimum led to significant changes in oxidant production and antioxidant defense. At 20 °C an increase in the intracellular H₂O₂ concentration and oxidized glutathione (GSSG) level was observed against a background of low levels of reduced glutathione (GSH) and decreased catalase and glutathione reductase (GOR) activities. The intracellular H₂O₂ and GSSG concentrations had minimal values at 30 and 37 °C, but rose again at 42 °C, suggesting that oxidative processes were intensified at high temperatures. An increase in temperature from 20 to 42 °C led to an elevation in the oxygen respiration rate and superoxide production; a 5-fold increase in the intracellular GSH concentration and in the GSH:GSSG ratio occurred simultaneously. Catalase HPI and GOR activities were elevated 4.4- and 1.5-fold, respectively. Prolonged exposure to sublethal temperatures facilitated an adaptation to subsequent oxidative stress produced by the addition of H₂O₂.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft” mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o + s_o) two-phase coexistence region below 8 ± 2 °C at ambient pressure. With increasing temperature, a l_o + l_d + s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o + l_d) immiscibility region is formed. Finally, above 48 ± 2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o + l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T = 37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10-35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r² ≥ 0.975), yielding median lethal times (LT₅₀s) of 14.3-150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT₅₀ of 110 (73-150) min, but could not grow at 10 or 15 °C. The mean LT₅₀ of five non-grasshopper isolates capable of growing at 10-35 °C was 16 (10-26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT₅₀s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48 °C with an inverse linearity (r² ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.     

Effect of temperature on isoproterenol-induced increases in left ventricular developed pressure

The goal of this project was to determine the effects of elevated cardiac temperature on preload-dependent and preload-independent regulation of left ventricular developed pressure (LVDP) in Langendorff-perfused, electrically paced (420 bpm), Sprague-Dawley rat hearts. LVDP responses to steady-state isoproterenol infusions (10⁻⁸ M) were determined at 37, 38, 39, and 40 °C. Preload-dependent LVDP was determined at 37 and 40 °C. Isoproterenol-induced LVDP and preload-dependent LVDP time controls were conducted in a separate group maintained at 37 °C. The percent increase in LVDP during isoproterenol infusion significantly decreased at 40 °C to 42±6 (SE), compared to 55±9, 55±6, and 53±7% at 37, 38, and 39 °C, respectively. No significant differences were observed in the percent increase in LVDP to isoproterenol among the corresponding time controls (50±6, 47±3, 56±4, and 56±5%). Preload-dependent LVDP decreased across the experimental protocol, but there were no cardiac temperature effects. These data indicate that β-adrenergic mediated contractility is not altered by moderate heating from normothermia but is compromised at very high temperatures (40 °C). Cardiac temperatures from 37 to 40 °C do not alter the inherent preload-dependent LVDP, indicating that the Frank–Starling relation is not directly affected within this temperature range.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

Thermal responses of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) acclimated at near-lethal temperatures, and the implications for climate change

The negative effects of climate alteration on coral reef fishes receive ever increasing attention; however, implications of rising sea temperatures on fishes inhabiting marine nursery environments are poorly understood. We used critical thermal methodology to quantify critical thermal maxima (CTmaxima) of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) captured from shallow seagrass nursery areas around Hoga Island, southeast Sulawesi, Indonesia. We tested the hypothesis that these distantly related fishes, when acclimated to cycling temperatures, would display higher CTmaxima than groups acclimated at constant temperatures. Groups of mullet acclimated to a constant temperature of 37 °C and temperature cycles of 35 to 39 °C or 37 to 41 °C displayed statistically similar mean CTmaxima of 44.7, 44.4 and 44.8 °C, respectively. Likewise, terapon acclimated at temperature cycles of 37 to 40 °C did not display a higher CTmaxima than fish acclimated at a constant temperature of 37 °C, with both acclimation groups' mean CTmaxima equal to 43.8 °C. Acclimation to higher cycling temperatures did not result in significant upper temperature tolerance acquisition for either species; however, mullet values were significantly higher than those seen in terapon (P < 0.0001). These data suggest that mullet and terapon will not suffer direct thermal effects should shallow nursery temperature increases be marginally higher than 1-2 °C above ~ 27 °C, and they provide evidence that the upper thermal tolerance of fishes inhabiting shallow seagrass and mangrove areas can approach the biokinetic limits for vertebrate life. Tropical marine fishes inhabiting fringing nursery environments may have the upper thermal tolerance necessary to endure substantial increases in sea temperatures.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K_m, but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25 °C but concealed at 37 °C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25 °C, but not at 37 °C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

Multigenerational analysis of temperature and salinity variability affects on metabolic rate,generation time,and acute thermal and salinity tolerance in Daphnia pulex

Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. Increasing levels of environmental stresses are known to induce the cellular stress response (CSR). Energy for the CSR may be provided by an elevated overall metabolic rate. However, if metabolic rate is constant or lower under elevated stress, energy for the CSR is taken from other physiological processes, such as growth or reproduction. This study investigated the examined energetic responses to the combination of temperature and salinity variability during a multigenerational exposure of partheogenetically reproducing Daphnia pulex. We raised D. pulex in an orthogonal combination of daily fluctuations in temperature (15, 15–25, 15–30 °C) and salinity (0, 0–2, 0–5). Initially metabolic rates were lower under all variable temperature and variable salinity treatments. By the 6th generation there was little metabolic variation among low and intermediate temperature and salinity treatments, but metabolic suppression persisted at the most extreme salinity. When grown in the control condition for the 6th generation, metabolic suppression was only observed in D. pulex from the most extreme condition (15–30 °C, 0–5 salinity). Generation time was influenced by acclimation temperature but not salinity and was quickest in specimens reared at 15–25 °C, likely due to Q₁₀ effects at temperatures closer to the optima for D. pulex, and slowest in specimens reared at 15–30 °C, which may have reflected elevated CSR. Acute tolerance to temperature (LT₅₀) and salinity (LC₅₀) were both highest in D. pulex acclimated to 15–30 °C and salinity 0. LT₅₀ and LC₅₀ increased with increasing salinity in specimens raised at 15 °C and 15–25 °C, but decreased with increasing salinity in specimens raised at 15–30 °C. Thus, increasing temperature confers cross-tolerance to salinity stress, but the directionality of synergistic effects of temperature and salinity depend on the degree of environmental variability. Overall, the results of our study suggest that temperature is a stronger determinant of metabolism, growth, and tolerance thresholds, and assessment of the ecological impacts of environmental change requires explicit information regarding the degree of environmental variability.     

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM.     

Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40 °C

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K₁k₊₂) and the maximum rate of dissociation (k₊₂) both increased with temperature, whereas 1/K₁ is unaffected by the change in temperature.     

The hydrolytic route to Co-porphyrin-doped SnO2 gas-sensing materials: Chemical study of Co-porphyrin versus Sn(IV) oxide interactions

SnO₂ and SnO₂ + Co-porphyrin solids were prepared from SnCl₄ in propanol and hydrolyzed to sol. Thermal behavior of samples obtained at 110 °C was studied in the 20-600 °C interval by thermal analysis coupled with mass spectrometry for identification of released species. The original samples maintain residual Sn-OR, Sn-OH and Sn-Cl groups up to 350 °C. The sample doped with 1% Co-porphyrin differs for a significant presence of residual Sn-Cl species, accounting for SnCl₄ release in the 300-340 °C range.¹¹⁹Sn solid state NMR analysis reveals disordered SnO₂ species in the sample heated at 250 °C and non-uniform SnO₆ units in the SnO₂ + Co-porphyrin sample at 110 °C, due to persistence of Sn-OR and Sn-OH groups. This complexity is lost at 250 °C. X-ray diffraction analysis confirms all these data. The sensing efficiency of these materials versus alcohols is ascribed to the presence of an open, incomplete SnO₂ structure, which is more pronounced in the Co-porphyrin-doped sample.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Ecofriendly degradation of sulfonated diazo dye C.I. Reactive Green 19A using Micrococcus glutamicus NCIM-2168

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l⁻¹) within 42 h at temperature 37 °C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.     

还原可降解聚磺酸甜菜碱型纳米水凝胶的制备及载药研究

目的:制备与表征还原可降解的聚磺酸甜菜碱型纳米水凝胶,利用该纳米递药系统包载阿霉素(DOX)并初步评价其抗肿瘤性能。方法:利用回流沉淀聚合的方法合成含二硫键的聚磺酸甜菜碱甲基丙烯酸酯(PSBMA)纳米水凝胶及不含二硫键的PSBMA纳米凝胶(nd-PSBMA);通过粒度仪和透射电镜考察两种纳米水凝胶的粒径、形态以及稳定性;通过考察谷胱甘肽(GSH)对纳米凝胶溶液相对浊度的影响以评价还原环境对两种纳米凝胶的还原可降解性;利用纳米凝胶包载阿霉素(DOX),考察载药凝胶在GSH中的释药行为,并初步评价其对A549肿瘤细胞的杀伤作用。结果:以N, N'-双丙烯酰胱胺为交联剂制备了含二硫键的PSBMA纳米凝胶,其粒径在180～200 nm;同时以N, N'-双丙烯酰胺为交联剂制备了不含二硫键的n-PSBMA纳米凝胶。两种纳米凝胶与小鼠血清共孵育7天水合粒径仍无明显变化,表明磺酸甜菜碱型纳米凝胶具有良好的抗蛋白吸附能力。此外,PSBMA纳米凝胶在GSH溶液中迅速地降解,且降解速度与GSH浓度呈正相关;而nd-PSBMA纳米凝胶在GSH溶液中几乎不降解。载DOX的PSBMA纳米凝胶可在GSH作用下快速的释放药物而载DOX的nd-PSBMA纳米凝胶在GSH作用下缓慢的释放药物;体外细胞实验显示空白纳米凝胶和载药nd-PSBMA对A549细胞无明显毒性作用,但载DOX的PSBMA纳米凝胶可高效地杀死A549肿瘤细胞,其药效与游离DOX相仿。结论:还原可降解的PSBMA纳米水凝胶有望成为智能型控释药物载体。     

Interaction of Na2B12H11SH with dimyristoyl phosphatidylcholine liposomes

Previous investigations have revealed that the boron cluster compound Na₂B₁₂H₁₁SH (BSH) is very potent in causing major structural rearrangements of and leakage from phosphatidylcholine liposomes. This somewhat unexpected finding is interesting from a fundamental point of view and may also constitute the basis of future important pharmaceutical/medical applications of BSH. In order to further explore the BSH-lipid interaction, we have studied the effects caused by BSH on dimyristoyl phosphatidylcholine (DMPC) liposomes.Cryo-transmission electron microscopy showed that BSH induces aggregation, membrane rupture and increasing wall thickness of the liposomes. Differential scanning calorimetry revealed a BSH dependent shift of the gel to liquid crystalline phase transition temperature of DMPC. The zeta potential of the liposomes decreases with increasing BSH concentrations, and an apparent dissociation constant of 0.23 mM was found.BSH caused leakage of liposome-encapsulated carboxyfluorescein; leakage was higher at 23 °C (near the phase transition temperature) than at 15 °C and 37 °C. It induced lipid mixing only at very high concentrations.