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The Survey of Organizational Research Climate (SOuRCe) is a new instrument that assesses dimensions of research integrity climate, including ethical leadership, socialization and communication processes, and policies, procedures, structures, and processes to address risks to research integrity. We present a descriptive analysis to characterize differences on the SOuRCe scales across departments, fields of study, and status categories (faculty, postdoctoral scholars, and graduate students) for 11,455 respondents from three research-intensive universities. Among the seven SOuRCe scales, variance explained by status and fields of study ranged from 7.6% (Advisor-Advisee Relations) to 16.2% (Integrity Norms). Department accounted for greater than 50% of the variance explained for each of the SOuRCe scales, ranging from 52.6% (Regulatory Quality) to 80.3% (Integrity Inhibitors). It is feasible to implement this instrument in large university settings across a broad range of fields, department types, and individual roles within academic units. Published baseline results provide initial data for institutions using the SOuRCe who wish to compare their own research integrity climates., (© The Author(s) 2014.)

Aflatoxins are highly toxic and carcinogenic fungal secondary metabolites. At least 18 enzyme activities are required for aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus. One of these enzymes, versicolorin B synthase (VBS), catalyzes bisfuran ring closure in versiconal hemiacetal (a reaction near the middle of the pathway) to form versicolorin B. This reaction is required for the subsequent activation to aflatoxin B1-8,9 epoxide, a highly reactive and toxic aflatoxin metabolite, and is important for aflatoxin toxicity. We analyzed the localization of VBS in the aflatoxin-producing strain A. parasiticus SU-1 grown on solid media using a colony fractionation technique developed previously. A highly specific polyclonal antibody, raised against a maltose-binding protein-VBS fusion protein synthesized in Escherichia coli, was used to detect VBS in SU-1 grown on a rich solid medium via immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold transmission electron microscopy (TEM). VBS was detected in both vegetative hyphae and in asexual developmental structures, called conidiophores. Western blot and CLSM analyses demonstrated the highest abundance of VBS in colony fraction S2 consisting of cells that had grown for 24-48 h; this fraction also contained the highest levels of newly developed conidiophores and the highest abundance of aflatoxin B1, consistent with VBS abundance. At the subcellular level, CLSM and TEM detected VBS distributed throughout the cytoplasm and concentrated in ring-like structures surrounding nuclei. It is uncertain whether enzymatically active VBS is present in either or both locations.

The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48-72 h; fraction 2, 24-48 h; fraction 3, 0-24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.

Vegetative (or heterokaryon) incompatibility is often characterized by cell death after anastomosis. In Neurospora crassa, partial diploid strains heterozygous for a single heterokaryon incompatibility (het) gene are viable, but grow at a significantly inhibited rate. Strains heterozygous for het-6 or het-c were examined microscopically for evidence of cell death; approximately 15% of cells randomly distributed within such colonies were dead or dying. Electron microscopy revealed extensive organelle degradation and plasmolysis. Ultimately, the cytoplasm fragmented into small membrane-bound bodies. Hyphal regrowth into dying cells from adjacent healthy cells was common. Ultrastructure and cell size measurements indicated no differences in death processes between incompatibility caused by het-6 and het-c. Linear growth rate was the only measured parameter which correlated with the observed macroscopic differences in colony morphology between het genes. The ultrastructural changes in dying cells were consistent with descriptions of apoptosis in plants and animals. However, designating vegetative incompatibility as apoptosis is premature without further study. Copyright 1998 Academic Press.

Broceeli (Brassika oleraceo L. var. capitata L.) leaf waxes were labelled with [2- 14 Cjacetate and examined during synthesis and deposition using microautoradiography. Waxes were viewed with light microscopy and scanning electron microscopy (SEM), including cryo-SEM. In microautoradiography, silver grains resulting from radioactive decay of labelled waxes seemed to be distributed randomly throughout the cuticle. This random distribution supports the hypothesis of diffusion or complex anastomosing pores as the mechanism of epicuriculur wax position. The hypothesis of wax-depositing pores running perpendicular to the leaf surface is not supported.

Agarose, agar, and gelatin were initially compared as encapsulation media for 3 structurally diverse particulate specimens: bacteria, yeast, and mitochondria. Agarose proved superior to both gelatin and agar for ease of handling and overall image quality (minimum background). All sample types exhibited high quality fixation and structural detail with no heat damage from the agarose medium. Based on this finding, we further characterized agarose encapsulation as affected by post-fixation, en bloc staining and resin type. Osmium tetroxide post-fixation, followed by en bloc uranyl acetate staining, could be performed without an increase in the electron density of the encapsulation medium. Agarose proved successful as an encapsulation medium regardless of the resin type or preparation protocol, thus providing flexibility in experimental design and excellent results over a range of variables.

Transmission electron microscopy (TEM) and energy dispersive X-ray microanalysis (EDS) were used to localize manganese from KMnO4, and bromine, as ultrastructural stains for lignin in an herbaceous plant. The Spookie cultivar of pumpkin is susceptible to infection by the fungus Colletotrichum lagenarium and served as a model system to compare the Br and KMnO4 techniques. Bromine was used in a fixation/staining procedure, and in separate experiments, KMnO4 was used as either a fixative or as a postsection stain. The technique for using bromine was modified from the woody plant procedure by adding a paraformaldehyde prefixation step. With the bromine procedure, cell walls were well-preserved, but the cytoplasm was heavily extracted. The KMnO4 procedures produced well-fixed cytoplasm, but with some staining artifacts. With all procedures, EDS dot mapping demonstrated lignin deposition in the cell walls specifically associated with sites of fungal infection. Lignin was also localized in secondary walls of tracheary elements, sites known to be highly lignified. The bromine procedure provided the most specific localization of lignin with a minimum of artifact. The specific applications of these stains provided data on the ultrastructural localization of lignin which contributed to the elucidation of its role in the interactions between pathogenic fungi in both their resistant and susceptible plant hosts.

In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.

We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosely attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source.

Electron microscopy has contributed a great deal to the field of mycology. Fungal ultrastructure has been, and continues to be, a key research element in the study of spore development and germination, host-pathogen interactions, nuclear behavior, and studies of subcellular organelles and organization linking structure and function. Since the earliest research in transmission electron microscopy in the 1950s, mycologists have kept pace with the developments in all areas of electron microscopy and have used them to great advantage in generating fine structural information on fungi. These recent developments include the use of scanning electron microscopy in the 1960s, X-ray microanalysis, cryopreservation and immunoelectron microscopy in the 1970s and 1980s. All of these techniques will continue to provide mycologists with the means to gain morphological and analytical data at the ultrastructural level.

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.