Expression of Drosophila Matrix Metalloproteinases in Cultured Cell Lines Alters Neural and Glial Cell Morphology.

Matrix metalloproteinases (MMPs) are zinc- and calcium- dependent endopeptidases that play pivotal roles in many biological processes. The expression of several MMPs in the central nervous system (CNS) have been shown to change in response to injury and various neurological/neurodegenerative disorders. While extracellular MMPs degrade the extracellular matrix (ECM) and regulate cell surface receptor signaling, the intracellular functions of MMPs or their roles in CNS disorders is unclear. Around 23 different MMPs are found in the human genome with overlapping function, making analysis of the intracellular role of human MMPs a daunting task. However, the fruit fly Drosophila melanogaster genome encodes only two MMPs: dMMP1 and dMMP2. To better understand the intracellular role of MMPs in the CNS, we expressed Green Fluorescent Protein (GFP)- tagged dMMPs in SH-SY5Y neuroblastoma cells and C6 glioblastoma cell lines. Lipofection of GFP-dMMPs in SH-SY5Y cells enhanced nuclear rupture and reduced cell viability (coupled with increased apoptosis) as compared to GFP alone. In non-liposomal transfection experiments, dMMP1 localizes to both the cytoplasm and the nucleus whereas dMMP2 had predominantly cytoplasmic localization in both neural and glial cell lines. Cytoplasmic localization demonstrated co-localization of dMMPs with cytoskeleton proteins which suggests a possible role of dMMPs in cell morphology. This was further supported by transient dMMP expression experiments that showed that dMMPs significantly increased neurite formation and length in neuronal cell lines. Inhibition of endogenous MMPs decreased neurite formation, length and ßIII Tubulin protein levels in differentiated SH-SY5Y cells. Further, transient expression experiments showed similar changes in glial cell morphology, wherein dMMP expression increased glial process formation and process length. Interestingly, C6 cells expressing dMMPs had a glia-like appearance, suggesting MMPs may be involved in intracellular glial differentiation. Inhibition or suppression of endogenous MMPs in C6 cells increased process formation, increased process length, modulated GFAP protein expression, and induced distinct glial-like phenotypes. Taken together, our results strongly support the intracellular role that dMMPs can play in apoptosis, cytoskeleton remodeling, and cell differentiation. Our studies further reinforce the use of Drosophila MMPs to dissect out the precise mechanisms whereby they exert their intracellular roles in CNS disorders.

Developing auditory-perceptual judgment reliability in otolaryngology residents.

OBJECTIVES: To determine how standard residency training and educational background affect otolaryngology resident auditory-perceptual judgments compared with inexperienced listeners. A secondary aim was to assess the impact of a brief training session on the reliability of judgments among otolaryngology residents. STUDY DESIGN: Mixed cross-sectional and cohort study. METHODS: Twenty adult dysphonic and four normal speaker samples were selected from a database. Fifteen listeners at various stages of residency in otolaryngology judged all samples for breathiness and roughness using visual analog scales. Fifteen inexperienced listeners judged the same samples. Subsequently, 12 otolaryngology residents underwent a brief training session. During training, listeners were provided definitions of rating dimensions and accuracy feedback while rating 20 novel (training) stimuli. The feedback included averaged responses from experienced clinicians who had previously evaluated the same speech samples. The residents then completed posttraining evaluations. RESULTS: As a group, otolaryngology resident listeners had significantly better interrater agreement for judgments of roughness compared with inexperienced listeners (P<0.05) and trended toward better interrater agreement for judgments of breathiness (P=0.058). Posttraining, no significant improvement in interrater agreement for judgments of roughness was observed; however, a significant improvement was found in interrater agreement of judgments of breathiness (P<0.05). CONCLUSIONS: Compared with inexperienced (untrained) listeners, otolaryngology resident listeners had better interrater agreement overall for judgments of dysphonia. A short training module was associated with improved reliability in resident evaluation of breathiness. Results have implications for improving and standardizing resident evaluation of dysphonia.

Relationship between nasal cavity volume changes and nasalance.

OBJECTIVES: The patency and volume of the nasal cavity affect the acoustic characteristics of the voice. The aim of this study was to investigate the effect of a nasal decongestant on nasal volumes and nasalance scores, and to determine the relationship between these measures. METHODS: Acoustic rhinometry and nasometry were performed in a group of 21 adult volunteers both prior to and following application of a nasal decongestant. The relationship between changes in nasalance scores and acoustic rhinometric parameters was investigated. RESULTS: After the application of nasal decongestant, statistically significant increases were observed in nasalance scores and in all of the acoustic rhinometric parameters assessed (i.e. minimal cross-sectional area, three cross-sectional areas, three volumes and total volume). However, no significant correlation was found between the changes in nasalance scores and acoustic rhinometric parameters. CONCLUSIONS: Nasal decongestion causes an increase in nasalance scores and nasal cavity volumes. However, the findings of this study indicate that changes in nasalance scores may result from factors other than nasal cavity volume changes.

Susceptibility of strawberries, blackberries, and cherries to Aspergillus mold growth and aflatoxin production.

The susceptibility of blackberries, cherries, and strawberries to Aspergillus growth and aflatoxin production has been examined. Three aflatoxigenic isolates of Aspergillus, A. flavus ATCC 15548 and NRRL 3251 as well as A. parasiticus NRRL 2999, were cultured on homogenates of the fruits for 14 days at 28 +/- 2 degrees C. Percent mycelial growth and spore infestation were determined each day with a calibrated grid. At day 14 each culture was frozen at -5 degrees C until aflatoxins were extracted with methylene chloride and water. Aflatoxins were separated by thin layer chromatography (TLC) with benzene-methanol-acetic acid (90 + 5 + 5). This extraction and solvent system provided satisfactory separations of the aflatoxins and was free of background interference on the TLC plates. Although all fruits served as substrates for both Aspergillus growth and aflatoxin production, cherries appeared to be a more favorable substrate than did blackberries, and the latter was more favorable than strawberries. Whereas A. flavus produced both B1 and G1 on all substrates, it yielded B2 and G2 only on cherries. Although A. parasiticus NRRL 2999 synthesized B1, B2, G1, and G2 on both blackberries and cherries, no aflatoxins were detected on strawberries. In contrast, A. flavus NRRL 3251 failed to produce detectable levels of aflatoxin on any substrate. All substrates supported both mycelial growth and subsequent sporulation with cherries greater than blackberries greater than strawberries.

Potential mold growth, aflatoxin production, and antimycotic activity of selected natural spices and herbs.

Ground spices and herbs are evaluated as substrates for mycelial growth, sporulation, and aflatoxin production. Three toxigenic strains of Aspergilli, A. flavus ATCC 15548, A. flavus NRRL 3251, and A. parasiticus NRRL 2999, were cultured on moist, commercially packaged herbs and spices. All substrates used were ground and included thyme, celery seed, oregano, cinnamon, ginger, caraway seed, clove, mustard, sesame seed, and rosemary leaves. Following inoculation of the natural materials in sterile bottles containing sterile water, the cultures were incubated 30 days at 23 +/- 4 degrees C. Not all strains of Aspergilli grew, sporulated, or produced toxins. There were definite strain differences and definite substrate differences for the variables evaluated. Sesame seed produced toxins B1, G1, and G2, with a mean of 167 ppm for 3 strains. A. flavus ATCC 15548 was the greatest overall toxin producer followed by A. parasiticus NRRL 2999 and A. flavus NRRL 3251. Ginger and rosemary leaves were also substantial producer-substrates. Mustard, caraway seed, and celery seed were judged as intermediate-producing substrates. Absolute antimycotic substrates were cinnamon and clove. Antiaflatoxigenic substrates were thyme and oregano. Mustard also may be antimycotic. Aflatoxins B1 and G1 were the more commonly found toxins.

Aflatoxigenic Potential for Aspergilli on sucrose substrates.

Sucrose concentrations of 3, 10, 20, and 30% in Czapek Dox broth served as the carbon source for growth, aflatoxin production, and sporulation for Aspergillus parasiticus NRRL 2999 and A. flavus NRRL 3557, 5862, and 5013. All cultures produced mycelial growth and sporulated in all sucrose concentrations during the 12-day growth period. The area of mycelial mat coverage per hour increased directly with increased sugar concentrations. The 20 and 30% sucrose concentrations inhibited mycelial growth for 5862. The 30% sucrose cultures of 3557 and 5862 failed to produce detectable levels of aflatoxins. All other isolates produced B1 and G1 in an approximately 4:1 ratio in all sucrose concentrations. Only 2999 was a substantial producer of aflatoxin in all 4 sucrose cultures, ranging from 72 to 96 micrograms/mL medium. A. flavus 5013 produced the most toxin, 144 and 126 micrograms/mL medium in the 10 and 20% sucrose cultures, respectively. The 10 and 20% sucrose cultures were most conducive to aflatoxin production. Since these sucrose levels correspond closely to the levels in many food and drink products, especially home-made products, care and attention should be taken to keep them free of aflatoxigenic spores. Extensive mycelial growth and sporulation, even in aflatoxigenic strains, do not necessarily result in comparably high aflatoxin levels. Routine extraction and quantitation procedures for aflatoxin were applicable and provided satisfactory results.

Growth, Sporulation and Aflatoxin Production by Aspergillus parasiticus on Strained Baby Foods.

The potential for aflatoxin production by Aspergillus parasiticus on strained baby food was evaluated. Four puréed foods were inoculated with the mold and cultured at 15 and 26 C in two series of experiments. The aflatoxigenic mold produced mycelia and sporulated at both temperatures. The foods ranked in mean total yield of aflatoxin (µg/g of substrate) in the following order: peas > squash > green beans > pears. The ranking held consistent for both temperatures. Aflatoxins B1 and G1 were produced in higher percentages than B2 and G2 in each food at both temperatures. At 26 C, total aflatoxin produced ranged from 8 to 71 µg/g of substrate, and at 15 C, the mean for the four foods was from 3 to 50 µg/g of substrate. Temperature and substrate were the primary variables which contributed to sporulation rate, toxin production and toxin ratios. Peas and squash should be considered primary and highly supportive substrates for aflatoxin production if conditions should arise for spores to contaminate the products either during or after processing. Absolute prevention of aflatoxigenic spore contamination in these foods studied is essential. An occasional testing of these foods for aflatoxin seems warranted. A lower temperature during aflatoxin formation decreased the total toxin formed, but did not prohibit aflatoxin occurrence. A lower temperature also tended to divert the type of toxin produced from B1 to the less dangerous G1 and G2. Aflatoxin would appear to be a problem in these foods only under rare and unusual circumstances in relation to processing and consumer usage. If such aflatoxigenic spore contamination should occur, the levels produced would be significant.

Aflatoxigenic potential of dried figs, apricots, pineapples, and raisins.

Spores from 3 aflatoxigenic strains were applied to the surface of dried figs, pineapple slices, apricots, and raisins. Half of the samples of each foodstuff was autoclaved (cooked) before inoculation. All 3 strains produced some toxin but not in all of the foodstuffs. No aflatoxins were found in any of the raisin cultures. The overall potential for toxin production in the dried fruit was apricot greater than fig greater than pineapple greater than raisin. Among the cooked substrates, the order was apricot greater than pineapple greater than fig=raisin. For the raw dried fruits, the ranking was fig greater than pineapple greater than apricot greater than or equal to raisin. Pineapple was considered the best broad spectrum substrate of the foodstuffs evaluated in the study.

Evaluation of potential for aflatoxin occurrence on celery, cauliflower, lettuce, and taro root inoculated with Aspergillus flavus and A. parasiticus.

Culture samples of lettuce, cauliflower, celery, and taro root (Colocasia esculenta) were assayed for the presence of aflatoxin after inoculation with Aspergillus flavus and A. parasiticus. Cultures of A. flavus produced both aflatoxins B1 and G1 on taro root, but produced by B1 on lettuce, cauliflower, and celery. For taro root, the percentage of aflatoxin G1 produced was considerably greater than that of B1. While A. parasiticus did produce mycelia and spores on the lettuce and taro root samples, there were not detectable levels of any aflatoxin produced. All the samples studied were successfully extracted and analyzed qualitatively and quantatively for the presence of aflatoxin by using official AOAC thin layer chromatographic procedures. There is sufficient evidence that Aspergilli can grow on some leafy produce and one strain produced aflatoxins.

Evaluating the inhibitory action of honey on fungal growth, sporulation, and aflatoxin production.

Unprocessed honey was inoculated with toxigenic strains of Aspergillus flavus NRRL 5862 and A. parasiticus NRRL 2999. The fungi grew and sporulated in varying amounts of honey diluted with water, but none of the cultures produced detectable levels of aflatoxin. Growth and subsequent sporulation were seen only in media containing up to and including 60% of honey. Media having 40% of honey showed growth and sporulation by day two. Neither species of Aspergillus produced toxins even in 10% honey. These results confirm our earlier observations that pure honey inhibited fungal growth and now even diluted honey seems capable of inhibiting toxin production or possibly neutralizing it. The general procedures recommended by the AOAC for extraction and thin layer chromatography were applied successfully in analyzing the honey substrate for aflatoxin.

Aflatoxin occurrence on raw and cooked york soybeans inoculated with three aspergillus isolates.

Raw and cooked soybean media were inoculated in separate experiments with Aspergillus flavus NRRL 3251, A. flavus ATCC 15548, and A. parasiticus NRRL 2999 isolates. The toatl quantity of aflatoxins produced and the percentage distribution of aflatoxins B1, B2, G1, and G2 varied with the state of the medium (raw or cooked) and with the fungal isolate used. Cooked soybean medium supported higher aflatoxin productions by A. flavus NRRL 3251 and A. parasiticus NRRL 2999 than did raw medium. Larger quantities of aflatoxins B1, B2, G1, and G2 were produced by A. flavus ATCC 15548 on raw soybean medium than were produced by any of the isolates on cooked medium. Application of these data is discussed briefly in relation to possible use of soybean media as a source of aflatoxin production.