RAS2 regulates growth and pathogenesis in Fusarium graminearum.

Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize. Diseases caused by F. graminearum are of particular concern because harvested grains frequently are contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but, when disrupted, caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the mitogen-activated protein (MAP) kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were reduced significantly in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway.

Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal.

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.

Difficult airway management in the neonate: a simple method of intubating through a laryngeal mask airway.

Tracheal intubation through a laryngeal mask airway is one option for securing an airway in the patient with a difficult airway. A variety of techniques and equipment have been used to stabilize the position of the tracheal tube while removing the laryngeal mask airway. We have shown that if a fibreoptic bronchoscope is used to place an tracheal tube through a laryngeal mask in neonates, additional equipment is not needed to remove the laryngeal mask airway without endangering tracheal tube placement. This is possible even in small neonates.

Green fluorescent protein as a reporter to monitor gene expression and food colonization by Aspergillus flavus.

Transformants of Aspergillus flavus containing the Aequorea victoria gfp gene fused to a viral promoter or the promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants.

Prevention of pediatric drowning and near-drowning: a survey of members of the American Academy of Pediatrics.

OBJECTIVE: To assess pediatricians' knowledge about the epidemiology of childhood drowning, their opinions and current practices regarding its prevention, and their interest in taking on more responsibility for its prevention. DESIGN: A self-administered questionnaire was mailed to 800 pediatricians in the United States, randomly selected from the American Academy of Pediatrics' approximately 18,000 full fellows. RESULTS: A total of 560 completed surveys were returned, a response rate of 70.1%. Although 85% of respondents believe it is the responsibility of pediatricians to become involved in community and/or legislative efforts to prevent childhood drowning, only 4.1% were involved in such efforts. Only a minority of respondents provided written materials and anticipatory guidance on drowning prevention to their patients. Women were more likely than men to discuss drowning prevention with their patients. Younger physicians were more likely than older physicians to discuss drowning prevention with their patients. Physicians who received formal education on drowning prevention during their pediatric residency training were more likely to provide written materials and anticipatory guidance on drowning prevention to their patients. However, only 17.9% of respondents received formal education on drowning prevention during their pediatric residency training. Seventy-four percent of all respondents felt that further education on the prevention of childhood drowning and near-drowning would be useful to them. CONCLUSION: Although drowning is the second leading cause of death by unintentional injury in the pediatric population (aged 0 to 19 years), most pediatricians do not routinely provide information to their patients, or to their patients' parents, on drowning prevention. IMPLICATION: Pediatricians have been effective child advocates in many areas of injury prevention. If the prevention of drowning is made a priority in pediatric practice, many more children's lives will be saved.

Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus.

The aflatoxin biosynthetic pathway regulatory gene, aflR, encodes a putative 47-kDa protein containing a zinc cluster DNA binding motif. It is required for the transcription of all of the characterized aflatoxin pathway genes in both Aspergillus flavus and Aspergillus parasiticus. The objective of this study was to examine the effects of aflR overexpression on temporal gene expression, aflatoxin production, and nitrate inhibition of aflatoxin biosynthesis in A. flavus. An inducible expression construct was made by fusing the coding region of aflR to the promoter region of the A. flavus adh1 gene. This construct was transformed into A. flavus 656-2 (FGSC A1010), a strain mutated at the aflR locus. Strain 656-2 containing the adh1(p)::aflR construct had induced transcription of two early aflatoxin pathway genes, nor-1 and pksA, and produced wild-type concentrations of aflatoxin in a temporal pattern similar to that of wild-type strains of A. flavus. Strains 656-2 and 86-10 (FGSC A1009) an aflatoxigenic strain, were transformed with a construct containing the constitutive promoter gpdA driving aflR. Transformants of these strains constitutively expressed aflR, fas-1A, pksA, nor-1, and omtA but did not constitutively produce aflatoxin. Strain 86-10 containing the gpdA(p)::aflR construct produced 50 times more aflatoxin than 86-10, but the temporal pattern of aflatoxin production was the same as for 86-10, and aflatoxin production was also induced by sucrose. The addition of 10 g of nitrate per liter to sucrose low salts medium inhibited aflatoxin production by both strain 86-10 and a transformant of 86-10 containing the gpdA(p)::aflR construct, indicating that nitrate inhibition of aflatoxin biosynthesis does not occur solely at the level of aflR transcription. These studies show that constitutive overexpression of the pathway transcriptional regulatory gene aflR leads to higher transcript accumulation of pathway genes and increased aflatoxin production but that the initiation of aflatoxin biosynthesis is not solely regulated by the transcriptional activities of the biosynthetic pathway.

Investigation of the devaluation interpretation of anticipatory negative contrast.

Rats suppress intake of an acceptable substance (e.g., 0.15% saccharin) when it is followed by a preferred substance (e.g., 32% sucrose) in once per day pairings. The role of a learned devaluation of the initial solution in suppressed intake (anticipatory negative contrast) was investigated. The findings included the following: (a) Flavors or odors as within-subject cues precluded the occurrence of anticipatory contrast, conditioning flavor and odor preferences instead, which appeared to antagonize suppressed intake. (b) Anticipatory contrast was obtained when within-subject context cues, temporal alternation cues, or drinking-spout cues were used. (c) Preference tests conducted with the spout cues showed that devaluation of the initial substance was not necessary for the occurrence of negative anticipatory contrast.

A beta-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. Current research is directed at the elimination of these compounds in important food sources. The objective of this research was to develop a method to study the induction and regulation of aflatoxin biosynthesis by examining the expression of one aflatoxin pathway gene, ver1. The promoter region of ver1 was fused to the beta-glucuronidase (GUS) gene (uidA) from Escherichia coli to form the reporter construct, GAP13. A. flavus 656-2 was transformed with this construct. Aflatoxin production, GUS activity, and transcript accumulation were determined in transformants after shifting the cultures from a nonconducive medium to a medium conducive to aflatoxin biosynthesis. Transformants harboring GAP13 displayed GUS expression only when aflatoxin was detected in culture. Further, the transcription of the uidA gene driven by the ver1 promoter followed the same profile as for the ver1 genes. The results show that the GAP13 construct may be useful as a genetic tool to study the induction of aflatoxin in situ and to identify substances that affect the expression of genes involved in aflatoxin biosynthesis. The utility of this construct to detect inducers of aflatoxin biosynthesis in maize kernels was tested in a bioassay. A heat-stable inducer of aflatoxin with a molecular size of less than 10 kDa was detected in extracts from maize kernels colonized by A. flavus.