3h Incubation Research Articles

Biodegradation of sulfoxaflor and photolysis of sulfoxaflor by ultraviolet radiation.

Sulfoxaflor (SUL, [N-[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl] ethyl]-λ4-sulfanylidene] cyanamide]) is a widely used systemic insecticide, and its residue has frequently been detected in the environment, posing a potential threat to the environment. In this study, Pseudaminobacter salicylatoxidans CGMCC 1.17248 rapidly converted SUL into X11719474 via a hydration pathway mediated by two nitrile hydratases (AnhA and AnhB). Extensive (96.4%) degradation of 0.83mmol/L SUL was achieved by P. salicylatoxidans CGMCC 1.17248 resting cells within 30min (half-life of SUL 6.4min). Cell immobilization by entrapment into calcium alginate remediated 82.8% of the SUL in 90min, and almost no SUL was observed in surface water after incubation for 3h. P. salicylatoxidans NHases AnhA and AnhB both hydrolyzed SUL to X11719474, although AnhA exhibited much better catalytic performance. The genome sequence of P. salicylatoxidans CGMCC 1.17248 revealed that this strain could efficiently eliminate nitrile-containing insecticides and adapt to harsh environments. We firstly found that UV irradiation transforms SUL to the derivatives X11719474 and X11721061, and the potential reaction pathways were proposed. These results further deepen our understanding of the mechanisms of SUL degradation as well as the environmental fate of SUL.

Differential expression of small heat shock proteins in the brain of broiler embryo; the effects of embryonic thermal manipulation.

Broilers are more vulnerable to high temperatures than mammals due to the feather cover, lack of sweat glands, fast growth and intensive breeding in commercial systems. Thermal stresses affect the function of various organs and change the expression profiles of hundreds of genes in the different tissues of broilers. Thermal manipulation (TM) during embryogenesis can increase heat tolerance in growing broilers. Small heat shock proteins (SHSPs) are a group of HSPs which participate in many cellular functions like response to different stressors. However, their role in the thermotolerance has not been fully elucidated. Ninety fertilized eggs were randomly divided into three groups (30 eggs/group; 10 eggs/replicate). Normal control (NC) eggs were incubated at 37.5°C throughout the incubation period whereas heat stress (HS) and cold stress (CS) groups were kept at 41°C and 33°C from 15 to 17th day of incubation for 3h each day, respectively. On day 20, samples from the cerebrums were harvested for histopathology and mRNA expression analyses of HSPB1, HSPB5, HSPB8, and HSPB9. There were no significant differences in survivability, defected embryos, hatchability, and body weight among treatments. TM had no major deleterious effects on the cerebral tissue except for mild degeneration in the HS group. HSPB1, HSPB5, HSPB8, and HSPB9 were expressed in the presence and absence of TM. All SHSP genes tested were downregulated in response to TM except for HSPB9 which was upregulated in the HS group. The highest change in gene expression due to TM observed for HSPB1. This study presents a broader understanding of mechanisms underlying response to TM in broilers. The results suggest that HSPB1, HSPB5, HSPB8, and HSPB9 are involved in thermotolerance in broilers and SHSPs could be involved in the gene expression profiling of TM. It may propose the use of nutritional supplements in the poultry industry to modulate SHSPs.

Heterologous expression and biochemical characterization of a thermostable endo-β-1,4-glucanase from Colletotrichum orchidophilum.

To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55-75°C and high thermostability (about 85% residual activity) at the temperature of 55°C after incubation for 3h. The highest activity of CoCel5A was detected when 100mM citric acid buffer (pH 4.0-5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100mM citric acid buffer (pH 3.0-6.0) at 4°C for 24h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-D-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70U/mg, 2.65mg/mL, and 75.67mL/mg/s, respectively; and 228.31U/mg, 2.06mg/mL, and 76.45mL/mg/s against β-D-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.

A Novel VIII Carboxylesterase with High Hydrolytic Activity Against Ampicillin from a Soil Metagenomic Library.

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50°C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3h at 60°C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C β-lactamases and family VIII esterases. As a consequence of its high sequence similarity with β-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

Development of Enteromorpha prolifera polysaccharide-based nanoparticles for delivery of curcumin to cancer cells

Polyelectrolyte complex nanoparticles were fabricated via electrostatic complexation between Enteromorpha prolifera polysaccharide (EP) and chitosan (CS). The monodisperse EP/CS NPs with negatively charge were prepared at pH4.0 and mixing ratio of 3.0:1.0. Curcumin loaded nanoparticles (CUR-NPs) showed spherical morphology with negatively charge of −16.27±0.97mV and average diameter range of 230 to 330nm. The results of spectroscopic analysis, XRD and DSC confirmed that there were hydrogen-bonding interaction and hydrophobic interaction between curcumin and EP/CS NPs. The CUR-NPs improved storage, thermal and photo stability of curcumin and exhibited sustained release of curcumin in vitro. Moreover, the CUR-NPs showed higher cellular uptake than free CUR with incubation for 3h by CLSM visualization and fluorescence quantitative assay. Furthermore, MTT assay results demonstrated that the CUR-NPs possessed good anticancer activity against B16F10 cells. Therefore, the EP-based nanoparticles are promising candidates for carriers in controllable hydrophobic anti-tumor drug delivery.

Thermal manipulation during embryogenesis improves certain semen parameters in layer breeder chicken during hot climatic conditions

Thermal manipulation during incubation has been shown to improve post hatch performance in poultry. The aim of the present experiment was to evaluate thermal manipulation on semen quality of roosters during hot climatic conditions. Eggs obtained after artificial insemination from Dahlem Red layer breeders were randomly divided into two groups control (C) and heat exposed (HE). C group eggs were incubated at 37.5°C throughout the incubation period while the HE group eggs were exposed to higher temperature 40.5°C from 15th to 17th day of incubation for 3h each day. The relative humidity was maintained at 65% in both the groups throughout incubation. The chicks hatched were reared separately under standard husbandry conditions. During high ambient temperature semen from roosters (45 weeks of age) was collected and evaluated for different gross parameters, sperm chromatin integrity and sperm HSP27 and HSP70 gene expression by real-time PCR. The seminal plasma was evaluated for lipid peroxidation, ferric ion reducing antioxidant power (FRAP), triiodothyronine (T3) and matrix metalloproteinase-2 (MMP-2) activity. The shed average Temperature Humidity Index (THI) during the experiment period was 78.55. The percent live sperm and FRAP level were significantly (P<0.05) higher and sperm gene expressions were significantly (P<0.05) lower in the HE group. No differences in other parameters were observed between the groups. Thus from the results it could be concluded that thermal manipulation during incubation improves certain semen parameters of roosters at high ambient temperature.

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ∼65nm and −49mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ∼130nm and −8mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3h with 150 and 450μM iron as BP-IONPs increased the cellular iron content from a low basal level of ∼50nmol iron mg−1 to 219±52 and 481±28nmol iron (mg protein)−1, respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.

Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells

Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 microM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100 microM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage.

Structural Studies on Casein Micelles of Human Milk: Dissociation of β-Casein of Different Phosphorylation Levels Induced by Cooling and Ethylenediaminetetraacetate

Information on the structure of human casein micelles has been obtained from dissociation of β-casein (CN). Two approaches were used: cooling at 4°C and addition of EDTA. An initial loss of about 80% of the protein optical density occurred upon cooling to 4°C. Dissociation was time dependent, and at ≥24h about 10% remained. However, mean size and voluminosity of micelles increased, as indicated by laser light scattering and viscosity measurements. This process was reversible, and 95% of the protein reentered the micelles upon incubation for 3h at 37°C. Upon cooling, amounts of nonphosphorylated β-CN increased, and singly phosphorylated β-CN levels were almost constant relative to the total β-CN in micelles. Upon addition of EDTA (0 to 5mM), the forms with three to five phosphates were the major dissociating constituents; EDTA that was added by dialysis produced similar results but at lower concentrations. These data suggest that, in the absence of significant amounts of αs1-CN, nonphosphorylated and singly phosphorylated human β-CN may form a framework, as proposed for αs1-CN for bovine milk, along with the colloidal calcium phosphate for the development of the final micelle structure by addition of the more highly phosphorylated forms. The results also indicate that human casein micelles have a less rigid structure than those of other species.

Glass transition and caking of spray‐dried lactose

Simple test methods to determine the onset temperatures of viscous flow and caking of spray‐dried amorphous lactose are described. The extent of viscous flow was measured as an increase in the density of lactose plugs within a cylindrical aluminium compaction apparatus after incubation for 3h at a specified temperature. Caking was characterized by an increase in the hardness of the lactose plugs formed. The onset temperature of viscous flow decreased with increasing water activity, Aw, and corresponded to the onset temperature of glass transition, Tg1. Glass transition temperatures were determined using both differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spin relaxation. The results suggest that elevation of the powder temperature above Tg1 promotes viscous flow and increases the potential for caking of amorphous food powders.

Effects of Scrotal Insulation on Viability Characteristics of Cryopreserved Bovine Semen

The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3-d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37°C for 3h. For assessment of results, collection days were grouped: period 1 (control) = d –6, –3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3,6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15,...39 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P<.05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P<.01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for both viability measurements, but only after incubation for 3h at 37°C postthaw (P<.05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation.

Multiple thyroid hormone binding sites on male rat liver nuclear matrices

Equilibrium binding of T3 to nuclear matrices isolated from male rat liver occurred after incubation for 3h at 20 degrees C. Two binding sites, having KD's of 6 and 95 nM, were revealed by Scatchard analysis. T3 and Triac competed for the binding of [125I]T3 to the high affinity site whereas only T3 competed for binding to the lower affinity site. Reverse T3 (rT3) did not compete for the binding of T3 to either class of binding sites. The binding sites were highly DNAse-sensitive, and less sensitive to protease treatment. The effect of binding of T3 to nuclear matrices by ATP, DTT and EDTA indicated that the sites are dissimilar to previously identified cytosolic binding sites. The higher affinity site resembles the T3 receptor in affinity and thyroid hormone specificity. The second site represents a new class of thyroid hormone binding sites. Its role in the regulation of thyroid hormone action warrants further investigation.

Milk Progesterone Enzyme Immunoassay: Modifications and a Field Trial for Pregnancy Detection in Dairy Cows1,2

A milk progesterone enzyme immunoassay was modified to shorten the antibody :antigen incubation time and tested in a field trial. Coupling the antibody to paper fibers at pH 7 increased the binding activity of the paper-antibody conjugate to allow incubation for 3h at room temperature with no significant loss of competitive binding of progesterone. The relationship between progesterone concentrations measured by the modified and original enzyme immunoassays was r=.94 (n=80 pairs). Milk samples (n=67) collected 21 d after artificial insemination were classified by the original and modified methods as being from pregnant or nonpregnant cows using both spectrophotometric and visual evaluations. Comparison to subsequent rectal palpation or return to estrus showed that the two methods were comparable. A field trial was conducted involving 622 cows and 40 producers using the modified enzyme immunoassay method. The overall field trial accuracy of the enzyme immunoassay in diagnosing the cow's reproductive status for a single sample on d 21 after breeding was 71% for correct diagnosis of pregnancy and 81% for correct diagnosis of nonpregnancy. It was concluded that the modified method is a valuable technique for rapid monitoring of milk progesterone concentrations.