Long-Term Survival of Shiga Toxin-Producing Escherichia coli O26, O111, and O157 in Bovine Feces

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25°C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10¹, 10³, and 10⁵ CFU/g. All three pathogens survived at 5 and 25°C for 1 to 4 weeks and at 15°C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25°C for 3 to 12 weeks, at 15°C for 1 to 18 weeks, and at 5°C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15°C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Thermal denaturation in acidic solutions of double-helical ribonucleic acid from virus-like particles found in Penicillium chrysogenum. A spectrophotometric study

1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of pK3.9 compared with pK2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε_(P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol<sup>−1</sup>·cm<sup>−1</sup> depending on pH, compared with 33501·mol<sup>−1</sup>·cm<sup>−1</sup> at pH7. In contrast ε<sub>(P)</sub> at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values.     

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties

Agro-industrial residues and cow dung were used as the substrate for the production of alkaline protease by Bacillus cereus strain AT. The bacterial strain Bacillus cereus strain AT produced a high level of protease using cow dung substrate (4813 ± 62 U g⁻¹). Physiological fermentation factors such as the incubation time (72 h), the pH (9), the moisture content (120%), and the inoculum level (6%) played a vital role in the enzyme bioprocess. The enzyme production improved with the supplementation of maltose and yeast extract as carbon and nitrogen sources, respectively. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymogram analysis of the purified protease indicated an estimated molecular mass of 46 kDa. The protease enzyme was stable over a temperature range of 40–50 °C and pH 6–9, with maximum activity at 50 °C and pH 8. Among the divalent ions tested, Ca²⁺, Na⁺ and Mg²⁺ showed activities of 107 ± 0.7%, 103.5 ± 1.3%, and 104.6 ± 0.9, respectively. The enzyme showed stability in the presence of surfactants such as sodium dodecyl sulfate and on various commercially available detergents. The crude enzyme effectively de-haired goat hides within 18 h of incubation at 30 °C. The enzymatic properties of this protease suggest its suitable application as an additive in detergent formulation and also in leather processing. Based on the laboratory results, the use of cow dung for producing and extracting enzyme is not cumbersome and is easy to scale up. Considering its cheap cost and availability, cow dung is an ideal substrate for enzyme bioprocess in an industrial point of view.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Herpesvirus salmonis: Characterization of a New Pathogen of Rainbow Trout

A new agent, provisionally designated Herpesvirus salmonis, was isolated from post-spawning rainbow trout (Salmo gairdneri) and studied primarily in the RTG-2 rainbow trout cell line. Infection of RTG-2 cells resulted in the formation of syncytia and Cowdry type A intranuclear inclusions. Replication occurred regularly at 5 and 10°C, but was inconsistent at 15°C, largely inhibited at 0°C, and completely inhibited at 20°C or higher. The virus was acid, heat, ether, and chloroform labile, but stable to freezing and thawing. It did not hemagglutinate. Viral DNA had a buoyant density of 1.709 g/cm³ and a guanine-cytosine value of 50%. Hexagonal nucleocapsids had a diameter of 90 nm and were first seen in nuclei at 36 h. Enveloped forms measured about 150 nm and occurred both cytoplasmically and extracellularly. At 10°C, a one-step growth culture required about 96 h; cell-associated virus peaked at about 10⁵ PFU/ml and exceeded released virus by a factor of about 10.     

Mortality,Temporary Sterilization,and Maternal Effects of Sublethal Heat in Bed Bugs

Adult bed bugs were exposed to the sublethal temperatures 34.0°C, 35.5°C, 37.0°C, 38.5°C, or 40.0°C for 3, 6, or 9 days. The two uppermost temperatures induced 100% mortality within 9 and 2 days, respectively, whereas 34.0°C had no observable effect. The intermediate temperatures interacted with time to induce a limited level of mortality but had distinct effects on fecundity, reflected by decreases in the number of eggs produced and hatching success. Adult fecundity remained low for up to 40 days after heat exposure, and the time until fertility was restored correlated with the temperature-sum experienced during heat exposure. Three or 6 days of parental exposure to 38.5°C significantly lowered their offspring’s feeding and moulting ability, which consequently led to a failure to continue beyond the third instar. Eggs that were deposited at 22.0°C before being exposed to 37.0°C for 3 or 6 days died, whereas eggs that were exposed to lower temperatures were not significantly affected. Eggs that were deposited during heat treatment exhibited high levels of mortality also at 34.0°C and 35.5°C. The observed negative effects of temperatures between 34.0°C and 40.0°C may be utilized in pest management, and sublethal temperature exposure ought to be further investigated as an additional tool to decimate or potentially eradicate bed bug populations. The effect of parental heat exposure on progeny demonstrates the importance of including maternal considerations when studying bed bug environmental stress reactions.     

Useful Host-Vector Systems in Bacillus stearothermophilus

We isolated a highly transformable thermophile, Bacillus stearothermophilus SIC1, which exhibited the following features. The growth temperature ranged from 45 to 65°C in L broth. The maximum cell concentration in 2L broth (2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2) was determined as an optical density at 660 nm of 7.8, and the generation time was 11 min at 60°C. Strain SIC1 was a prototroph and was transformed by the protoplast procedure not only with repB plasmids (high-copy-number plasmids such as pTB913 and pUB110) but also with repA plasmids (low-copy-number plasmids such as pTB53). Transformation efficiencies with repB and repA plasmids were about 2 × 10⁶ to 5 × 10⁶ and 5 × 10⁴ transformants per μg of DNA, respectively. The transformant carrying plasmid pTB913Y/K could grow at 63°C in the presence of kanamycin. The regeneration frequency of protoplasts was 60%, and only 1 day was needed for regeneration at 55°C.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Electric Field-Induced Changes in Lipids Investigated by Modulated Excitation FTIR Spectroscopy

The effect of electric fields on dry oriented multibilayers of dimyristoylphosphatidylcholine (DMPC) was investigated by transmission Fourier transform infrared electric field modulated excitation (E-ME) spectroscopy. A periodic rectangular electric potential (0–150 V, 1.25 Hz, 28.4°C ± 0.2°C) was applied across the sample. To discriminate electric field-induced effects from possible temperature-induced effects resulting from a current flow (<1 pA) across the sample, corresponding temperature-modulated excitation (T-ME) measurements within the temperature uncertainty limits of ±0.2°C at 28.4°C were performed. T-ME induced reversible gauche defects in the hydrocarbon chains, whereas E-ME resulted in reversible compression of dry DMPC bilayers. Periodic variation of the tilt angle of the hydrocarbon chains is suggested. The degree of absorbance modulation in the CH-stretching region was found to be in the order of 1:700, corresponding to a variation of the bilayer thickness of Δz = 0.0054 nm. Using a series connection of capacitors as equivalent circuit of the cell resulted in E = (1.2 ± 0.7) × 10⁷ V/m for the electric field in DMPC. Young's elasticity modulus of DMPC could be calculated to be E = 2.2 × 10⁶ Pa ± 1.8 × 10⁶ Pa, which is in good agreement with published data obtained by electric field-dependent capacitance measurements.     

Characterization of EV-2, a Virus Isolated from European Eels (Anguilla anguilla) with Stomatopapilloma

A virus designated EV-2 has been isolated from external tumor tissue and internal organs of European eels (Anguilla anguilla) with stomatopapilloma. It contains RNA and is ether, acid, and temperature labile above 4°C, and concentrated preparations agglutinate chicken and sheep erythrocytes. The addition of actinomycin D during the first 2.75 h of infection inhibits viral replication. As determined in sucrose gradients, the buoyant density of the virus is 1.19 g/cm³. EV-2 has a moderately pleomorphic spherical morphology; its diameter ranges from 80 to 140 nm. The virion has narrow, regularly spaced surface projections about 10 nm long. Replication in FHM cells at 15°C shows new infectivity appearing at 10 h postinfection and reaching a plateau at 20 h. Cytopathic effects consist of cell fusion, syncytia, and irregularly rounded cell masses. Viral antigen was detected in the cytoplasm of infected cells by specific immunofluorescence.     

Newly Identified Thermostable Esterase from Sulfobacillus acidophilus: Properties and Performance in Phthalate Ester Degradation

EstS1, a newly identified thermostable esterase from Sulfobacillus acidophilus DSM10332, was heterologously expressed in Escherichia coli and shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant (k_cat/K_m) of the enzyme for p-nitrophenyl butyrate was as high as 6,770 mM⁻¹ s⁻¹. The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Levels of Endogenous Indole-3-acetic Acid in Achenes of Rosa rugosa during Dormancy Release and Germination

Indole-3-acetic acid (IAA) in highly purified extracts of rose achenes (Rosa rugosa var rubra) was quantified by means of ion-pair reversephase high performance liquid chromatography with spectrofluorimetric detection. Changes in IAA content were determined during a 14-week 4°C stratification, which leads to dormancy breakage, and during subsequent germination at 17°C. IAA was also determined in achenes stratified in parallel at 17°C, which does not induce release from dormancy. IAA decreased during the first 2 weeks of stratification both at 4°C and at 17°C. IAA remained low during the remaining 12 weeks of stratification at 4°C, whereas it continued to decrease in achenes kept at 17°C. An immediate increase in IAA during germination was followed by transients in the IAA level. The results suggest that IAA is without a regulating role in dormancy release although it seems to be involved in the germination process.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Nutrigenomics approach elucidates health-promoting effects of high vegetable intake in lean and obese men

We aimed to explore whether vegetable consumption according to guidelines has beneficial health effects determined with classical biomarkers and nutrigenomics technologies. Fifteen lean (age 36 ± 7 years; BMI 23.4 ± 1.7 kg m⁻²) and 17 obese (age 40 ± 6 years; BMI 30.3 ± 2.4 kg m⁻²) men consumed 50- or 200-g vegetables for 4 weeks in a randomized, crossover trial. Afterward, all subjects underwent 4 weeks of energy restriction (60 % of normal energy intake). Despite the limited weight loss of 1.7 ± 2.4 kg for the lean and 2.1 ± 1.9 kg for the obese due to energy restriction, beneficial health effects were found, including lower total cholesterol, LDL cholesterol and HbA1c concentrations. The high vegetable intake resulted in increased levels of plasma amino acid metabolites, decreased levels of 9-HODE and prostaglandin D3 and decreased levels of ASAT and ALP compared to low vegetable intake. Adipose tissue gene expression changes in response to vegetable intake were identified, and sets of selected genes were submitted to network analysis. The network of inflammation genes illustrated a central role for NFkB in (adipose tissue) modulation of inflammation by increased vegetable intake, in lean as well as obese subjects. In obese subjects, high vegetable intake also resulted in changes related to energy metabolism, adhesion and inflammation. By inclusion of sensitive omics technologies and comparing the changes induced by high vegetable intake with changes induced by energy restriction, it has been shown that part of vegetables’ health benefits are mediated by changes in energy metabolism, inflammatory processes and oxidative stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0343-9) contains supplementary material, which is available to authorized users.     

Detection of γH2AX foci in mouse normal brain and brain tumor after boron neutron capture therapy

Aim

In this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.

Background

Boron neutron capture therapy (BNCT) is a particle radiation therapy in combination of thermal neutron irradiation and boron compound that specifically accumulates in the tumor. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of extremely high linear energy transfer (LET) radiation and therefore have marked biological effects. High LET radiation causes severe DNA damage, DNA DSBs. As the high LET radiation induces complex DNA double strand breaks (DSBs), large proportions of DSBs are considered to remain unrepaired in comparison with exposure to sparsely ionizing radiation.

Materials and methods

We analyzed the number of γH2AX foci by immunohistochemistry 30 min or 24 h after neutron irradiation.

Results

In both normal brain and brain tumor, γH2AX foci induced by ¹⁰B(n,α)⁷Li reaction remained 24 h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24 h after irradiation in these tissues.

Conclusion

DSBs produced by ¹⁰B(n,α)⁷Li reaction are supposed to be too complex to repair for cells in normal brain and brain tumor tissue within 24 h. These DSBs would be more difficult to repair than those by γ-ray. Excellent anti-tumor effect of BNCT may result from these unrepaired DSBs induced by ¹⁰B(n,α)⁷Li reaction.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. × V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [³H]GA₄ (of high specific activity, 4.81 × 10¹⁰ becquerel per millimole) for 48 hours at 26°C. Chilling had little effect on the total amount of free [³H]GA-like metabolites formed during incubation at 26°C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [³H] metabolites formed at 26°C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA₁₂ aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [³H]GA₄ treatment at 26°C, reduced the uptake of [³H] GA₄ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26°C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).     

Detection of Phosphatidylcholine-Coated Gold Nanoparticles in Orthotopic Pancreatic Adenocarcinoma using Hyperspectral Imaging

Nanoparticle uptake and distribution to solid tumors are limited by reticuloendothelial system systemic filtering and transport limitations induced by irregular intra-tumoral vascularization. Although vascular enhanced permeability and retention can aid targeting, high interstitial fluid pressure and dense extracellular matrix may hinder local penetration. Extravascular diffusivity depends upon nanoparticle size, surface modifications, and tissue vascularization. Gold nanoparticles functionalized with biologically-compatible layers may achieve improved uptake and distribution while enabling cytotoxicity through synergistic combination of chemotherapy and thermal ablation. Evaluation of nanoparticle uptake in vivo remains difficult, as detection methods are limited. We employ hyperspectral imaging of histology sections to analyze uptake and distribution of phosphatidylcholine-coated citrate gold nanoparticles (CGN) and silica-gold nanoshells (SGN) after tail-vein injection in mice bearing orthotopic pancreatic adenocarcinoma. For CGN, the liver and tumor showed 26.5±8.2 and 23.3±4.1 particles/100μm² within 10μm from the nearest source and few nanoparticles beyond 50μm, respectively. The spleen had 35.5±9.3 particles/100μm² within 10μm with penetration also limited to 50μm. For SGN, the liver showed 31.1±4.1 particles/100μm² within 10μm of the nearest source with penetration hindered beyond 30μm. The spleen and tumor showed uptake of 22.1±6.2 and 15.8±6.1 particles/100μm² within 10μm, respectively, with penetration similarly hindered. CGH average concentration (nanoparticles/μm²) was 1.09±0.14 in the liver, 0.74±0.12 in the spleen, and 0.43±0.07 in the tumor. SGN average concentration (nanoparticles/μm²) was 0.43±0.07 in the liver, 0.30±0.06 in the spleen, and 0.20±0.04 in the tumor. Hyperspectral imaging of histology sections enables analysis of phosphatidylcholine-coated gold-based nanoparticles in pancreatic tumors with the goal to improve nanotherapeutic efficacy.