Alkaline dissolution of starch facilitated by microwave heating for analysis by size-exclusion chromatography

A rapid, straightforward starch dissolution method was developed to facilitate analysis of starch by size-exclusion chromatrography (SEC). Soft wheat starch was dispersed in 6 M urea and 1 M KOH and subjected to heating for 35, 45, or 60 s in a microwave oven as a potential means of starch dissolution. An increase in microwave heating time enhanced starch solubility but resulted in a steady decrease in amylopectin peak area and a consequent, artifactual increase in the amylose peak area due to molecular degradation. However, microwave heating for 35 s afforded a reasonable separation of starch fractions by SEC while minimizing molecular degradation of starch in comparison to a traditional starch dissolution procedure. This procedure provides a fast (<30 min), simple, and reproducible starch dissolution method for preparation of starches for SEC analysis and represents the first successful report of direct dissolution of granular starch via microwave heating.     

Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal soft wheat starch

This work fractionated waxy and normal wheat starches into highly purified A- and B-type granule fractions, which were representative of native granule populations within parent native wheat starches, to accurately assess starch characteristics and properties of the two granule types. Wheat starch A- and B-type granules possessed different morphologies, granule specific surface area measurements, compositions, relative crystallinities, amylopectin branch chain distributions, and physical properties (swelling, gelatinization, and pasting behaviors). Within a genotype, total and apparent amylose contents were greater for A-type granules, while lipid-complexed amylose and phospholipid contents were greater for B-type granules. B-type (relative to A-type) granules within a given genotype possessed a greater abundance of short amylopectin branch chains (DP_n < 13) and a lesser proportion of intermediate (DP_n 13–33) and long (DP_n > 33) branch chains, contributing to their lower relative crystallinities. Variation in amylose and phospholipid characteristics appeared to account for observed differences in swelling, gelatinization, and pasting properties between waxy and normal wheat starch fractions of a common granule type. However, starch granule swelling and gelatinization property differences between A- and B-type granules within a given genotype were most consistently explained by their differential amylopectin chain-length distributions.     

Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.     

Topical KINOSTAT™ ameliorates the clinical development and progression of cataracts in dogs with diabetes mellitus

Objective To determine whether topical administration of the aldose reductase inhibitor Kinostat™ can ameliorate the onset or progression of cataracts in dogs with naturally occurring diabetes mellitus (DM). Materials and Methods A randomized, prospective, double‐masked placebo control pilot study was conducted with 40 dogs newly diagnosed with DM with no or minimal lens changes. Twenty‐eight dogs received Kinostat™ and 12 dogs received placebo. Procedures Owners administered the agent into both eyes three times daily for 1 year and compliance was monitored with log sheets. Complete ophthalmic examinations were performed on dilated eyes at the time of enrollment and 1, 2, 3, 6, and 12 months into treatment. Cataract severity was assessed on a scale of 0–3. At 12 months, full bloodwork, including HbA1C and blood Kinostat^TM levels were performed. Results After 12 months of treatment, the cataract score in the placebo group significantly increased with seven dogs (14 eyes) developing mature cataracts, two dogs (4 eyes) developing cortical opacities, and one dog (2 eyes) developing equatorial vacuoles with mild punctate cortical opacities. In contrast, the cataract score in the Kinostat^TM treated dogs was significantly less with seven developing anterior equatorial vacuoles, two developing incipient anterior cortical cataracts, and four developing mature cataracts. In fact, the cataract scores of the Kinostat^TM group at 12 months did not significantly increase from the score at the time of enrollment. The HbA1C values between the two groups after 12 months of treatment were similar, and no blood levels of Kinostat^TM were found in any enrolled dog. Conclusion The onset and/or progression of cataracts in dogs with DM can be significantly delayed by topical administration of Kinostat™.     

Channels within soft wheat starch A- and B-type granules

The nature of channels within wheat starch granules was investigated using scanning electron and confocal laser scanning microscopy. A-type granules stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA, protein-specific probe) revealed a network of radially oriented, channel-like protein structures similar to those previously reported. However, treatment of the same starch granules with methanolic merbromin (fluorescent dye) solution, which is used to highlight external granule surfaces (including those of channels) under non-swelling conditions, revealed few, if any, channels extending into the granule interior. This discrepancy suggested that channels within wheat starch granules were filled at least in part with protein. Removal of protein with protease facilitated greater access of methanolic merbromin to channels and/or cavities for both granule types. For A-type starch granules, relatively large channels were observed in the equatorial groove region, while finer channels originated from other regions of the granule. This work reports the first visualization of B-type granule channels, which most frequently occurred as less-defined voids (as opposed to the fine, discrete channels of A-type granules) extending to granule surfaces. Channels of A- and B-type starch granules appeared to facilitate transfer of chemical reagent into the granule matrix, though this effect was aided by granule swelling (hydration) and/or removal of channel-associated protein.     

Smoke-derived taint in wine: the release of smoke-derived volatile phenols during fermentation of Merlot juice following grapevine exposure to smoke

The release of smoke-derived volatile phenols during the fermentation of Merlot grapes, following grapevine exposure to smoke, has been investigated. The concentrations of guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, and eugenol were determined by gas chromatography-mass spectrometry and found to increase throughout the winemaking process. Only trace levels (< or = 1 microg/L) of guaiacol and 4-methylguaiacol could be detected in free run juice derived from the fruit of smoked vines; the highest levels, 388 microg/L and 93 microg/L, respectively, were observed in the finished wine. Control wine (derived from fruit of unsmoked vines) contained 4 microg/L guaiacol, with the volatile phenols either not detected or detected at only trace levels (< or = 1 microg/L) throughout fermentation. The role of enzyme and acid catalyzed hydrolysis reactions in releasing smoke-derived volatile compounds was also investigated. The volatile phenols were released from smoked free run juice by strong acid hydrolysis (pH 1.0) and enzyme (beta-glucosidase) hydrolysis, but not mild acid hydrolysis (juice pH 3.2-3.7). Guaiacol was again the most abundant smoke-derived phenol, present at 431 microg/L and 325 microg/L in strong acid and enzyme hydrolysates, respectively. Only trace levels of each phenol could be detected in each control hydrolysate. This study demonstrates the potential for under-estimation of smoke taint in fruit and juice samples; the implications for the assessment of smoke taint and quantification of volatile phenols are discussed.     

Spatial and temporal variability in CH4 and N2O fluxes from a Scottish ombrotrophic peatland: Implications for modelling and up-scaling

Peatlands typically exhibit significant spatial heterogeneity which can lead to large uncertainties when catchment scale greenhouse gas fluxes are extrapolated from chamber measurements (generally <1 m²). Here we examined the underlying environmental and vegetation characteristics which led to within-site variability in both CH₄ and N₂O emissions and the importance of such variability in up-scaling. We also consider within-site variation in the controls of temporal dynamics. Net annual emissions (and coefficients of variation) for CH₄ and N₂O were 1.06 kg ha⁻¹ y⁻¹ (300%) and 0.02 kg ha⁻¹ y⁻¹ (410%), respectively. The riparian zone was a significant CH₄ hotspot contributing ∼12% of the total catchment emissions whilst covering only ∼0.5% of the catchment area. In contrast to many other studies we found smaller CH₄ emissions and greater uptake in chambers containing either sedges or rushes. We also found clear differences in the drivers of temporal CH₄ dynamics across the site, e.g. water table was important only in chambers which did not contain aerenchymous plants. We suggest that depending on the heterogeneity of the site, flux models could be improved by incorporating a number of spatially distinct sub-models, rather than a single model parameterized using whole-catchment averages.     

Development of a new management strategy for the control of root-knot nematodes (Meloidogyne spp) in organic vegetable production

The nematophagous fungus, Pochonia chlamydosporia (Goddard) Zare & Gams, has been investigated as a potential biological control agent for use in integrated pest management strategies for Meloidogyne incognita (Kof & White) Chitwood in vegetable crops. The release of the fungus as a biological control agent requires a diagnostic method to monitor its spread in the environment and to gain knowledge of its ecology. Only molecular methods are sufficiently discriminating to enable the detection of specific isolates of fungi in soil. A method to extract DNA from soil was developed to increase the efficacy of PCR-based diagnostic tests that use specific primers. A selected isolate of P chlamydosporia var catenulata was applied at densities similar to those that occur naturally in nematode-suppressive soils. The fungus significantly reduced nematode infestations in soil following a tomato crop, in a strategy that combined the use of the fungus with crop rotation. The survival of the fungus in soil was also examined in controlled conditions in which it remained in soil in densities significantly greater than its original application rate for at least 5 months. Hence, it seems that populations of this fungus may be built up in soil and have significant effects on the regulation of root-knot nematode populations.