Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Laboratory Study of Slurry Seal Coats, HR-185, Revised, 1978

Extensive programmed laboratory tests involving some 400 asphalt emulsion slurry seals (AESS) were conducted. Thirteen aggregates including nine Iowa sources, a quartzite, a synthetic aggregate (Haydite), a limestone stone from Nebraska, and a Chat aggregate from Kansas were tested in combination with four emulsions and two mineral fillers, resulting in a total of 40 material combinations. A number of meetings were held with the Iowa DOT engineers and 12 state highway departments that have had successful slurry seal experiences and records, and several slurry seal contractors and material and equipment suppliers were contacted. Asphalt emulsion slurry seal development, uses, characteristics, tests, and design methods were thoroughly reviewed in conjunction with Iowa's experiences through these meetings and discussions and through a literature search (covering some 140 articles and 12 state highway department specifications). It was found that, while asphalt emulsion slurry seals (when properly designed and constructed) can economically improve the quality and extend the life of existing pavement surface, experiences with them had been mixed due to the many material, slurry, and construction variables that affect their design, construction, and performance. The report discusses those variables identified during the course of the project and makes recommendations concerning design procedures, design criteria, specifications and the means of evaluating them.

Incorporation and washout of n-3 PUFA after high dose intravenous and oral supplementation in healthy volunteers.

BACKGROUND & AIMS: Although the physiological effects of n-3 polyunsaturated fatty acids (n-3PUFA) are generally thought to require several weeks of exposure to allow their incorporation into plasma membranes, intravenous (IV) n-3PUFA attenuate the cardiovascular and neuroendocrine response to stress within 3 h. Whether oral n-3 PUFA exert similar early effects remains unknown. OBJECTIVE: To assess whether acute IV or short term oral n-3PUFA administration reproduces the metabolic effects of long term oral supplements during exercise, and how it relates to their incorporation into platelets and red blood cells (RBC) membranes. DESIGN: Prospective single center open label study in 8 healthy subjects receiving a 3-h infusion of 0.6 g/kg body weight n-3PUFA emulsion, followed one week later by an oral administration of 0.6 g/kg over 3 consecutive days. Maximal power output (cycling exercise), maximal heart rate (HR), blood lactate at exhaustion, and platelet function were measured at baseline and after IV or 3-day oral supplementation; platelet and RBC membrane composition were assessed until 15 days after n-3PUFA administration. RESULTS: Both IV and oral n-3PUFA significantly decreased maximal HR (-6% and -5%), maximal power output (-10%) and peak blood lactate (-47% and -52%) Platelet function tests were unchanged. The EPA and DHA membrane contents of RBC and platelets increased significantly, but only to 1.7-1.9% of fatty acid content. CONCLUSION: The cardiovascular and metabolic effects of n-3 PUFA during exercise occur already within 1-3 days of exposure, and may be unrelated to changes in membranes composition. Effects occur within hours of administration and are unrelated to lipid membrane composition. Trial registered at clinicaltrials.gov as NCT00516178.

Field Performance and Evaluation of Slurry Seals, HR-195, 1980

As part of the overall research program of evaluating asphalt emulsion slurry seal as a pavement maintenance material, 31 duplicate 500-ft test sections were constructed on U.S. 6 between Adel and Waukee in Dallas County during September and October of 1978. These test sections included combinations of eight aggregates, two gradings, three asphalt emulsions, two mineral fillers, and a range of emulsion contents determined by laboratory mix designs. The emulsion contents of the test sections varied from 10.3% for Section 7A (Ferguson coarse) to 32.9% for Section 31A (lightweight aggregate). The post-construction performance evaluation of the test sections, consisting primarily of the friction tests and surface appearance observations, was conducted at different time intervals up to 24 months after construction. At the 24-month final evaluation, most of the test sections had carried a total of 1.4 million vehicles.

Drying rates of Rubi grapes submitted to chemical pretreatments for raisin production

The objective of this work was to determine the most appropriated chemical treatment to be used to dry grapes cv. Rubi for raisin production. Drying curves for convective drying with air at 50ºC, in a tray drier, were obtained for grapes submitted to chemical pretreatments with different concentrations of potassium carbonate and olive oil, and different dipping times, according to factorial designs. Convective drying curves were also obtained for grapes pretreated in aqueous suspensions of soybean lecithin, at varied lecithin concentrations and dipping times. Page model was adjusted to the experimental drying curves, and the calculated drying times showed that the best pretreatment consisted in dipping grapes for 2 minutes in a 5% olive oil and 6% K2CO3 emulsion, at 50ºC, which resulted in a drying time close to that of the pretreatment with 2.5% of olive oil, but with a lower consumption of this substance. In addition, the immersion of grapes in an aqueous suspension of 2% soy lecithin, at 50ºC, for 5 minutes, resulted in a total drying time slightly higher than the most effective pretreatment.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

Analysis of the contribution of the beta-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae.

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Asphalt Binders for Thin Maintenance Surfaces, TR-435, 2003

Asphalt is used as a binder for thin maintenance surface (TMS) applications because of two key properties, it is waterproof and it adheres relatively well to the aggregate. Since asphalt is too stiff at room temperature to apply to the road surface, it is usually applied as either a cutback asphalt or an asphalt emulsion. The asphalt emulsions can be further divided into high float emulsions, cationic emulsions or polymer-modified binders, which are emulsions with polymers added to them. These types of binders are discussed further below.

Examination of Curing Criteria for Cold In-Place Recycling, TR-553, 2008

Cold In-Place Recycling (CIR) has been used widely in rehabilitating the rural highways because it improves a long-term pavement performance. A CIR layer is normally covered by a hot mix asphalt (HMA) overlay in order to protect it from water ingress and traffic abrasion and obtain the required pavement structure and texture. Curing is the term currently used for the period of time that a CIR layer should remain exposed to drying conditions before an HMA overlay is placed. The industry standard for curing time is 10 days to 14 days or a maximum moisture content of 1.5 percent, which appear to be very conservative. When the exposed CIR layer is required to carry traffic for many weeks before the wearing surface is placed, it increases the risk of a premature failure in both CIR layer and overlay. This study was performed to explore technically sound ways to identify minimum in-place CIR properties necessary to permit placement of the HMA overlay. To represent the curing process of CIR pavement in the field construction, three different laboratory curing procedures were examined: 1) uncovered, 2) semi-covered and 3) covered specimens. The indirect tensile strength of specimens in all three curing conditions did not increase during an early stage of curing but increased during a later stage of curing usually when the moisture content falls below 1.5%. Dynamic modulus and flow number increased as curing time increased and moisture contents decreased. For the same curing time, CIR-foam specimens exhibited the higher tensile strength and less moisture content than CIR-emulsion. The laboratory test results concluded that the method of curing temperature and length of the curing period significantly affect the properties of the CIR mixtures. The moisture loss index was developed to predict the moisture condition in the field and, in the future, this index be calibrated with the measurements of temperature and moisture of a CIR layer in the field.

Examination of Curing Criteria for Cold In-Place Recycling Phase 2: Measuring Temperature, Moisture, Deflection and Distress from CIR Test Section, TR-590, 2009

The previous research performed laboratory experiments to measure the impacts of the curing on the indirect tensile strength of both CIR-foam and CIR-emulsion mixtures. However, a fundamental question was raised during the previous research regarding a relationship between the field moisture content and the laboratory moisture content. Therefore, during this research, both temperature and moisture conditions were measured in the field by embedding the sensors at a midpoint and a bottom of the CIR layer. The main objectives of the research are to: (1) measure the moisture levels throughout a CIR layer and (2) develop a moisture loss index to determine the optimum curing time of CIR layer before HMA overlay. To develop a set of moisture loss indices, the moisture contents and temperatures of CIR-foam and CIR-emulsion layers were monitored for five months. Based on the limited field experiment, the following conclusions are derived: 1. The moisture content of the CIR layer can be monitored accurately using the capacitance type moisture sensor. 2. The moisture loss index for CIR layers is a viable tool in determining the optimum timing for an overlay without measuring actual moisture contents. 3. The modulus back-calculated based on the deflection measured by FWD seemed to be in a good agreement with the stiffness measured by geo-gauge. 4. The geo-gauge should be considered for measuring the stiffness of CIR layer that can be used to determine the timing of an overlay. 5. The stiffness of CIR-foam layer increased as a curing time increased and it seemed to be more influenced by a temperature than moisture content. The developed sets of moisture loss indices based on the field measurements will help pavement engineers determine an optimum timing of an overlay without continually measuring moisture conditions in the field using a nuclear gauge.

Examination of Curing Criteria for Cold In-Place Recycling (Phase 3: Calibration of Moisture Loss Indices and Development of Stiffness Gain Model), TR-609, 2011

In the previous study, moisture loss indices were developed based on the field measurements from one CIR-foam and one CIR-emulsion construction sites. To calibrate these moisture loss indices, additional CIR construction sites were monitored using embedded moisture and temperature sensors. In addition, to determine the optimum timing of an HMA overlay on the CIR layer, the potential of using the stiffness of CIR layer measured by geo-gauge instead of the moisture measurement by a nuclear gauge was explored. Based on the monitoring the moisture and stiffness from seven CIR project sites, the following conclusions are derived: 1. In some cases, the in-situ stiffness remained constant and, in other cases, despite some rainfalls, stiffness of the CIR layers steadily increased during the curing time. 2. The stiffness measured by geo-gauge was affected by a significant amount of rainfall. 3. The moisture indices developed for CIR sites can be used for predicting moisture level in a typical CIR project. The initial moisture content and temperature were the most significant factors in predicting the future moisture content in the CIR layer. 4. The stiffness of a CIR layer is an extremely useful tool for contractors to use for timing their HMA overlay. To determine the optimal timing of an HMA overlay, it is recommended that the moisture loss index should be used in conjunction with the stiffness of the CIR layer.

Bond Contribution to Whitetopping Performance on Low Volume Roads, Construction Report, HR-341, 1993

This research was initiated in 1991 as a part of a whitetopping project to study the effectiveness of various techniques to enhance bond strength between a new Portland cement concrete (PCC) overlay and an existing asphalt cement concrete (ACC) pavement surface. A 1,676 m (5,500 ft) section of county road R16 in Dallas County, Iowa was divided into 12 test sections. The various techniques used to enhance bond were power brooming, power brooming with air blast, milling, cement and water grout, and emulsion tack coat. As a part of these bonding techniques, two pavement thicknesses were placed; two different concrete proportions were used; and two sections were planed to a uniform cross-slope.

Field Evaluation of Cold In-Place Recycling of Asphalt Concrete, HR-303, Construction Report, 1990

There are still many vintage portland cement concrete (PCC) pavements, 18 ft wide (5.4 m), dating back to pre-World War II era in use today. Successive overlays have been placed to cover joints and to improve rideability. The average thickness of the existing asphalt cement concrete (ACC) along route E66 in Tama County, Iowa, was 6.13 in. (15.6 cm). The rehabilitation strategy called for widening the base using the top 3 in. (7.6 cm) of the existing ACC by a recycling process involving cold milling and mixing with additional emulsion/rejuvenator. The material was then placed into a widening trench and compacted to match the level of the milled surface. This project was undertaken to develop a rehabilitation methodology to widen these older pavements economically and to have a finished surface capable of carrying traffic with little or no additional work.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.