Phylogenetic analysis of Monascus and new species from honey, pollen and nests of stingless bees.

The genus Monascus was described by van Tieghem (1884) to accommodate M. ruber and M. mucoroides, two species with non-ostiolate ascomata. Species delimitation in the genus is still mainly based on phenotypic characters, and taxonomic studies that include sequence data are limited. The genus is of economic importance. Species are used in fermented Asian foods as food colourants (e.g. 'red rice' (ang-kak, angka)) and found as spoilage organisms, and recently Monascus was found to be essential in the lifecycle of stingless bees. In this study, a polyphasic approach was applied combining morphological characters, ITS, LSU, ß-tubulin, calmodulin and RNA polymerase II second largest subunit sequences and extrolite data, to delimit species and to study phylogenetic relationships in Monascus. Furthermore, 30 Monascus isolates from honey, pollen and nests of stingless bees in Brazil were included. Based on this polyphasic approach, the genus Monascus is resolved in nine species, including three new species associated with stingless bees (M. flavipigmentosus sp. nov., M. mellicola sp. nov., M. recifensis sp. nov., M. argentinensis, M. floridanus, M. lunisporas, M. pallens, M. purpureus, M. ruber), and split in two new sections (section Floridani sect. nov., section Rubri sect. nov.). Phylogenetic analysis showed that the xerophile Monascus eremophilus does not belong in Monascus and monophyly in Monascus is restored with the transfer of M. eremophilus to Penicillium (P. eremophilum comb. nov.). A list of accepted and excluded Monascus and Basipetospora species is given, together with information on (ex-)types cultures and barcode sequence data.

Effect of germicidal UVC light on fungi isolated from grapes and raisins.

AIMS: To examine how UVC affects the different genera of fungi commonly isolated from grapes, with the aim of understanding changes in mycobiota during grape ripening and possible applications for preventing grape decay during storage. METHODS AND RESULTS: Spores of Aspergillus carbonarius, Aspergillus niger, Cladosporium herbarum, Penicillium janthinellum and Alternaria alternata (between 100-250 spores/plate agar) were UVC irradiated for 0 (control), 10, 20, 30, 60, 300 and 600 s. Plates were incubated at 25 degrees C and colonies were counted daily up to 7 days. Alternaria alternata and Aspergillus carbonarius were the most resistant fungi. Conidial germination in these species was reduced by approx. 25% after 10 s of exposure, compared with greater than 70% reduction for the remaining species tested. Penicillium janthinellum spores were the most susceptible at this wavelength. UVC exposures of 300 s prevented growth of all isolates studied, except for Alternaria alternata. CONCLUSIONS: UVC irradiation plays a major role in selecting for particular fungi that dominate the mycobiota of drying grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC irradiation of harvested grapes could prevent germination of contaminant fungi during storage or further dehydration.

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans.

AIMS: To determine the incidence and severity of infection by ochratoxin A (OA)-producing fungi in Vietnamese green coffee beans. METHODS AND RESULTS: Aspergillus carbonarius, A. niger and yellow Aspergilli (A. ochraceus and related species in section Circumdati) were isolated by direct plating of surface-disinfected Robusta (65 samples) and Arabica (11 samples) coffee beans from southern and central Vietnam. Significantly, more Robusta than Arabica beans were infected by fungi. Aspergillus niger infected 89% of Robusta beans, whereas A. carbonarius and yellow Aspergilli each infected 12-14% of beans. OA was not produced by A. niger (98 isolates) or A. ochraceus (77 isolates), but was detected in 110 of 113 isolates of A. carbonarius, 10 isolates of A. westerdijkiae and one isolate of A. steynii. The maximum OA observed in samples severely infected with toxigenic species was 1.8 microg kg(-1); however, no relationship between extent of infection and OA contamination was observed. CONCLUSIONS: Aspergillus niger is the dominant species infecting Vietnamese coffee beans, yet A. carbonarius is the likely source of OA contamination. SIGNIFICANCE AND IMPACT OF STUDY: Vietnamese green coffee beans were more severely infected with fungi than the levels reported for beans from other parts of the world, yet OA contamination appears to be infrequent.

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia.

AIMS: The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. CONCLUSIONS: OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. SIGNIFICANCE AND IMPACT OF THE STUDY: Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.

Australian research on ochratoxigenic fungi and ochratoxin A.

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.

Effects of water activity and temperature on the survival of Aspergillus carbonarius spores in vitro.

AIMS: To determine the effect of water activity (a(w)) and temperature on the survival of Aspergillus carbonarius spores. METHODS AND RESULTS: Spores of A. carbonarius were dried onto filter membranes. These filters were held at 1.0, 0.9, 0.8, 0.6 and 0.4 a(w) and at 1, 15, 25 and 37 degrees C for up to 618 d. At intervals, spores were recovered from filters and assessed for viability by enumeration on dichloran rose bengal chloramphenicol agar. Survival and subsequent growth of spores was prolonged at low temperatures and at a(w) below 0.6. Above 15 degrees C, 0.6-0.9 a(w) were often more deleterious than 1.0. However, at 1 degrees C and 1.0 a(w), spores lost viability more rapidly than at lower a(w). CONCLUSIONS: Increased incidence of black Aspergillus spp. in dry soils and from grapes in dry conditions may result partly from prolonged survival of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Delineating the direct effect of a(w) and temperature on survival of A. carbonarius spores may aid in understanding the incidence of this ochratoxin A-producing species in vineyard soils and on grapes.

Congenital cystic adenomatoid malformation of the lung: indications and timing of surgery.

We review a single surgeon and surgical centre's experience with congenital cystic adenomatoid malformation of the lung (CCAML) in relation to clinical spectrum, operative experience, and postoperative course. A retrospective hospital record review was done on surgically treated cases of CCAML over a 10-year period, focusing on number with antenatal diagnosis, spectrum of postnatal presentation, type of surgery performed, and outcome. Forty-seven patients from birth to 14 years of age underwent surgery for CCAML. Antenatal diagnosis (ante) was made in 30 cases. Of these, 10 became symptomatic before surgery. Six of the 17 postnatally-diagnosed (pnd) cases were an asymptomatic incidental finding. Overall, 16 were symptomatic in the 1st year of life, and five were symptomatic beyond 1 year of age. Symptoms varied from respiratory distress (seven ante, six pnd) to chronic cough (three, and recurrent chest infection (three ante, two pnd). All preoperative diagnoses were confirmed with chest CT. Most patients (25) were operated on before 3 months of age. Eleven were operated on in the first 2 weeks of life as emergency surgery for respiratory distress. The most common lobe involved was the right upper lobe (16), and lobectomy was performed in 42 cases, segmentectomy in four, and pneumonectomy in one. Seventeen cases were extubated immediately postoperatively; 29 required postoperative ventilation overnight, and nine needed more prolonged ventilation. Early postoperative complications included pneumothorax (two), pleural effusion (one), and chylous effusion (one). Late complications included recurrence in three cases (all segmentectomy), who then subsequently underwent lobectomy. There was one death from respiratory failure. Because there is an increasing trend in the detection of asymptomatic antenatally-diagnosed CCAML, consideration of early surgical excision to prevent complications is suggested by our series. CT scanning is mandatory for postnatal evaluation because chest x-ray could be normal. Safe elective excision after 3 months is supported by our low morbidity and less need for postoperative ventilation. Lobectomy is the procedure of choice to prevent recurrence.

Web-based education in family medicine predoctoral programs.

BACKGROUND AND OBJECTIVES: New technologies and widespread Internet access make Web-based education feasible for family medicine predoctoral programs. Offering educational activities and resources through the Web can support community-based education and improve communication. We assessed the status of Web-based education nationally and explored the interest and opportunities for collaboration. METHODS: A survey assessed the inclusion of Web-based educational methods in family medicine predoctoral programs and school-wide programs, the level of national interest in collaborative development, and common obstacles to progress. The survey was mailed to all US family medicine predoctoral directors. RESULTS: The response rate was 61%. Results showed nearly universal use of e-mail and Web pages. The most common educational use of the Internet was posting text information. One third of the responding programs used the Web for evaluation. Barriers to development of Web-based educational programs are faculty time and funding. Most respondents were interested in collaborative Web development and would value a national, Society of Teachers of Family Medicine-based network for this purpose. CONCLUSIONS: Web-based educational activities are commonly offered by family medicine programs. To realize the full potential of Web-based education, collaborative development of new methods and materials will be needed to overcome the limiting factors of faculty time and funding.

Evaluating students on an interdisciplinary primary care clerkship at the Pennsylvania State University College of Medicine.

With funding from The Robert Wood Johnson Foundation's Generalist Physician Initiative, the Pennsylvania State University College of Medicine created a community-based primary care clerkship in general pediatrics, general internal medicine, and family and community medicine, in which third-year students spend a month in a small town, rural area, or urban underserved medical community. In addition to linking students with preceptors who would teach the clinical skills essential to primary care practice, the medical school set out to teach and to evaluate knowledge, attitudes, and behaviors unique to primary care. This paper describes the three-part teaching tool/evaluation developed to address (1) a student's recognition of the characteristics of primary care (learning objectives assignment), (2) a student's ability to appreciate the multiple nonmedical factors influencing a patient's health and experience of illness (family project), and (3) a student's ability to solve clinical problems (clinical reasoning examination). The authors describe how these evaluation methods are linked with the clerkship's goals and objectives and how they yield a richer portrait of the student's performance than the traditional preceptor's evaluation alone can provide. They also discuss the relationship between students' performances on the primary care clerkship and their performances in other clinical clerkships. Similar clinical experiences in primary care should focus on features unique to primary care medicine in both teaching and evaluation.

The standardized family: an innovation in primary care education at the University of Massachusetts.

In 1995 as part of The Robert Wood Johnson Foundation's Generalist Physician Initiative, the University of Massachusetts (UMass) Medical School developed the "standardized family" as a new model for teaching the essential elements of primary care in a core curriculum format outside of the clinical setting. Using this model, a hypothetical family unit (the "McQ Family") serves as the focus for case-based clinical problem solving. This paper describes the standardized family model and provides two years of evaluation outcomes such as curriculum assessments, student performance outcomes, and correlation with external measures of clinical performance to support the effectiveness of this educational model. It discusses the transferability of the standardized family model from UMass Medical School to Pennsylvania State University College of Medicine and uses Pennsylvania State's adaptation (the "Hershey-Penn Family") to illustrate how the standardized family can be customized to integrate a core curriculum into a three-year longitudinal primary care program. The authors suggest that the standardized family model has the potential to meet a broad range of primary care teaching needs at other institutions.

7-Hydroperoxycholesterol and its products in oxidized low density lipoprotein and human atherosclerotic plaque.

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (> or = 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (< 0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse-phase method utilizing chemiluminescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 beta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.

Inhibition of protein synthesis by reduced K+ in cultured endothelial cells.

Total protein synthesis (as precipitable [3H] leucine incorporation) was determined under various experimental conditions to examine the relationship between cellular K+ and protein synthesis in human umbilical vein endothelial cells (HUVEC). We found that under normal external K+ concentration (5 mM), total protein synthesis was inhibited by cycloheximide and ouabain, with estimated values of IC50 of 0.41 microM and 0.60 mM, respectively. Cellular K+ concentrations were determined (102 +/- 4 mM for control cells) and found to be significantly increased (P < 0.01) by high external K+ (25 mM) and significantly decreased (P < 0.001) by low external K+ (0.5 mM) as well as by ouabain (2 mM). Under high external K+, total protein synthesis and the inhibitory responses of cycloheximide and ouabain were not altered. On the contrary, cellular K+ and protein synthesis were both further reduced by about half (P < 0.001) under low external K+. While ouabain further reduced cellular K+ by half (P < 0.001), protein synthesis was only slightly reduced (P < 0.05) under low external K+ and thus the relative reduction on protein synthesis was much less than that for cellular K+. These results indicate that while elevated intracellular K+ did not alter protein synthesis, reduced intracellular K+ correlated with a reduced cellular protein synthesis in HUVEC such that K+ may play a permissible role in the regulation of protein synthesis.