Nondestructive and Rapid Screening of Aflatoxin-Contaminated Single Peanut Kernels Using Field-Portable Spectroscopy Instruments (FT-IR and Raman).

A nondestructive and rapid classification approach was developed for identifying aflatoxin-contaminated single peanut kernels using field-portable vibrational spectroscopy instruments (FT-IR and Raman). Single peanut kernels were either spiked with an aflatoxin solution (30 ppb-400 ppb) or hexane (control), and their spectra were collected via Raman and FT-IR. An uHPLC-MS/MS approach was used to verify the spiking accuracy via determining actual aflatoxin content on the surface of randomly selected peanut samples. Supervised classification using soft independent modeling of class analogies (SIMCA) showed better discrimination between aflatoxin-contaminated (30 ppb-400 ppb) and control peanuts with FT-IR compared with Raman, predicting the external validation samples with 100% accuracy. The accuracy, sensitivity, and specificity of SIMCA models generated with the portable FT-IR device outperformed the methods in other destructive studies reported in the literature, using a variety of vibrational spectroscopy benchtop systems. The discriminating power analysis showed that the bands corresponded to the C=C stretching vibrations of the ring structures of aflatoxins were most significant in explaining the variance in the model, which were also reported for Aspergillus-infected brown rice samples. Field-deployable vibrational spectroscopy devices can enable in situ identification of aflatoxin-contaminated peanuts to assure regulatory compliance as well as cost savings in the production of peanut products.

FRET-based sensor for CaMKII activity (FRESCA): A useful tool for assessing CaMKII activity in response to Ca2+ oscillations in live cells.

Ca2+ oscillations and consequent Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation are required for embryogenesis, as well as neuronal, immunological, and cardiac signaling. Fertilization directly results in Ca2+ oscillations, but the resultant pattern of CaMKII activity remains largely unclear. To address this gap, we first employed the one existing biosensor for CaMKII activation. This sensor, Camui, comprises CaMKIIα and therefore solely reports on the activation of this CaMKII variant. Additionally, to detect the activity of all endogenous CaMKII variants simultaneously, we constructed a substrate-based sensor for CaMKII activity, FRESCA (FRET-based sensor for CaMKII activity). To examine the differential responses of the Camui and FRESCA sensors, we used several approaches to stimulate Ca2+ release in mouse eggs, including addition of phospholipase Cζ cRNA, which mimics natural fertilization. We found that the Camui response is delayed or terminates earlier than the FRESCA response. FRESCA enables assessment of endogenous CaMKII activity in real-time by both fertilization and artificial reagents, such as Sr2+, which also leads to CaMKII activation. FRESCA's broad utility will be important for optimizing artificial CaMKII activation for clinical use to manage infertility. Moreover, FRESCA provides a new view on CaMKII activity, and its application in additional biological systems may reveal new signaling paradigms in eggs, as well as in neurons, cardiomyocytes, immune cells, and other CaMKII-expressing cells.

A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal.

The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal.

Validation of the Ceredigion Youth Screening Tool.

Evidence suggests that only a small minority of youth offenders will continue their behaviour in the longer term and largely independent of any interventions they may receive (Bateman, 2011; Haines & Case, 2015). Hence, "screening out" this larger low-risk cohort could have a positive impact upon the individual through a reduction in stigmatisation/labelling and free up resources for higher risk clients. This article outlines development of the Ceredigion Youth Screening Tool (CYSTEM)-developed and tested to address the two facets of criminality and vulnerability-closely aligned to the eight key risk indicators identified in the Risk-Needs-Responsivity (R-N-R) literature (Andrews & Bonta, 2010). Initial results with two cohorts of 372 young people indicate good convergent and discriminative validity in screening out the lowest level referrals, while also identifying 90% of potential future offenders. More importantly, CYSTEM is able to screen out approximately 35% of the low-risk offenders that are unlikely to require formal evaluation and/or intervention. It is suggested that the streamlining of this process using CYSTEM reduces demand on staff time and decreases the stigmatisation of young people referred for minor offences. Potential improvements to the tool and future developments in statistical risk prediction are also discussed.

Anthocyanin condensed forms do not affect color or chemical stability of purple corn pericarp extracts stored under different pHs.

Purple corn is rich in anthocyanins, some of which are condensed with flavanols. The aim was to determine the impact of anthocyanin condensed forms extracted from purple corn pericarp on color and chemical stability at different pHs compared with the complete extract, and an extract without condensed forms. Extracts were dissolved at pH values ranging from 2.0 to 6.0 and stored for 12weeks at 22°C. Color stability of anthocyanins decreased as the pH increased. Slight color differences were observed throughout time at pH 2 (ΔE from 0.2 to 3.6). After 12weeks, pH 6 caused substantial changes in color (ΔE=17.7 to 47.5); and reduced the predicted half-life of total anthocyanins (ranging from 1.8 to 3weeks), compared to pH 2 (44.6 to 60.7weeks). Condensed forms had degradation kinetics similar to monomeric anthocyanins. Purple corn pericarp pigments can be used in acid beverages with an acceptable shelf-life.

Survey of Anthocyanin Composition and Concentration in Diverse Maize Germplasms.

Increasing consumer demand for natural ingredients in foods and beverages justifies investigations into more economic sources of natural colorants. In this study, 398 genetically diverse pigmented accessions of maize were analyzed using HPLC to characterize the diversity of anthocyanin composition and concentration in maize germplasm. One hundred and sixty-seven accessions were identified that could produce anthocyanins in the kernel pericarp or aleurone and were classified into compositional categories. Anthocyanin content was highest in pericarp-pigmented accessions with flavanol-anthocyanin condensed forms, similar to the Andean Maíz Morado landraces. A selected subset of accessions exhibited high broad-sense heritability estimates for anthocyanin production, indicating this trait can be manipulated through breeding. This study represents the most comprehensive screening of pigmented maize lines to date and will provide information to plant breeders looking to develop anthocyanin-rich maize hybrids as an economic source of natural colorants in foods and beverages.

Temperature dependency of shelf and thermal stabilities of anthocyanins from corn distillers' dried grains with solubles in different ethanol extracts and a commercially available beverage.

The objective was to determine the shelf and thermal stabilities of anthocyanins from distillers' dried grains with solubles (DDGS) extracted with different ethanol concentrations as well as a semi-purified Maiz Morado (purple corn) anthocyanin extract added to a commercially available beverage. Storage for 6 weeks of DDGS showed an overall reduction of anthocyanins from 6.8 to 73.7%. In DDGS, an ethanol increase from 0 to 25% resulted in less sensitivity of anthocyanin to temperature changes. Acylation resulted in faster degradation and higher reaction rate constants than their corresponding non-acylated forms. Anthocyanin changes were accompanied by an overall increase in lightness and a decrease in redness. Storage of beverage for 12 weeks at 4 °C resulted in a 25.5% reduction of anthocyanin. Results have important implications in selecting colored corn as an economical source of food colorants.

Processing method and corn cultivar affected anthocyanin concentration from dried distillers grains with solubles.

Anthocyanins are water-soluble pigments with health benefits and potential use as food colorants. The objectives of this work were to (1) determine optimum parameters for the extraction of anthocyanins from dried distillers grain with solubles (DDGS), (2) develop a method of anthocyanin extraction from DDGS, (3) quantify and identify the extracted anthocyanins, and (4) determine the effect of processing methods and corn cultivars on anthocyanin concentration. DDGS samples were prepared from purple (PC) and dark (DC) corn and processed using conventional enzymes (C) and granular starch hydrolyzing enzymes (GC). Three independent variables (ethanol concentration (0, 12.5, and 25%); liquid-to-solid ratio (30:1, 40:1, 50:1 mL/g); and extraction temperature (4, 22, and 40 °C)) and two dependent variables (anthocyanin concentration and a-value (redness)) were used. Results showed that dark corn DDGS gave anthocyanin concentration higher than that of purple corn. The GC process showed total anthocyanin concentration higher than that of the conventional method of DDGS production. The maximum anthocyanin concentration was obtained at 12.5% ethanol, 40:1 liquid-to-solid ratio, and 22 °C for C-PC [321.0 ± 37.3 µg cyanidin-3 glucoside (C3G) equivalent/g DDGS]. For GC-PC, 25% ethanol, 30:1 liquid-to-solid ratio, and 22 °C gave 741.4 ± 12.8 µg C3G equivalent/g DDGS. For GC-DC, 12.5% ethanol, 40:1 liquid-to-solid ratio, and 40 °C extraction gave 1573.4 ± 84.0 µg C3G equivalent/g DDGS. LC/MS-MS analysis showed that the major anthocyanins were cyanidin-3-glucoside, cyanidin-3-(6″-malonyl) glucoside, and peonidin-3-(6″malonyl) glucoside. In conclusion, anthocyanin extraction from colored corn DDGS can be optimized using 12.5% ethanol, 40:1 mL/g ratio, and 22 °C.

Two types of transgenic lines for doxycycline-inducible, cell-specific gene expression in zebrafish ultraviolet cone photoreceptors.

Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On system to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTA(flag)). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16 h of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTA(flag)) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2a(IntraWT) was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2a(IntraWT)), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTA(flag)) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease.

Color and chemical stability of a variety of anthocyanins and ascorbic acid in solution and powder forms.

The color and chemical stabilities of six anthocyanins, including cyanidin 3-glucoside, highly purified and present in semipurified extracts (also containing other anthocyanins) from grape pomace, purple corn, and black rice, were determined in combination with ascorbic acid in solutions at differing pH values (3.0 and 4.0) and temperatures (6-40 °C) and lyophilized powders at different relative humidities (43-98% RH). Color and chemical changes were analyzed using CIELAB measurements and HPLC, respectively. In liquids, stability was inversely related to increasing pH and temperature; for powders, stability was inversely related to RH. The mutual destruction of anthocyanins and ascorbic acid in solution was confirmed, with unexpected new findings showing no significant stabilizing/destabilizing effect based upon anthocyanin structure, including differing flavylium core (three types) and type of acylation (two aliphatic, one cinnamic acid), or final extract purity.

Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.

Ribeye a-mCherry fusion protein: a novel tool for labeling synaptic ribbons of the hair cell.

Synaptic ribbons are presynaptic cytomatrices that are required for efficient transfer of auditory information from hair cells to the central nervous system. In the hair cell, each electron-dense ribbon tethers numerous synaptic vesicles by fine filaments. The ribbon generally resides juxtaposed to the active zone plasma membrane. A dearth of appropriate tools to visualize the ribbon synapse has limited our knowledge of its development. Here we present the design and implementation of a method to visualize synaptic ribbons in hair cells. This scheme uses a tagged version of the protein Ribeye a, which is specific to ribbons. We generate the DNA construct Tg(pvalb3b:ribeye a-mCherry) to transgenically express the fusion protein Ribeye a-mCherry in zebrafish hair cells. The fusion protein localizes to the basolateral surface of the hair cell with a pattern similar to that of a hair cell labeled with an antiserum that recognizes ribeye proteins. Moreover, using this antiserum to label transgenics that express Ribeye a-mCherry, we demonstrate that the fusion protein and antibody-associated fluorescent signals overlap. In addition, ribbons labeled with the fusion protein are proximal to afferent nerve endings. Finally, the fusion protein labels hair-cell ribbons of zebrafish at different developmental time points. These findings indicate that the fusion protein is an effective tool to label ribbons in live and fixed hair cells, which will make it useful in the study of ribbon synapse development and to characterize zebrafish mutants with defects in synapse formation.