Prolonged cadmium exposure alters benign uterine fibroid cell behavior, extracellular matrix components, and TGFB signaling.

The heavy metal Cadmium (Cd), a widespread environmental contaminant, poses serious hazards to human health and is considered a metallohormone and carcinogen. In women with uterine fibroids, there is a significant association between blood Cd levels and increased fibroid tumor size. The aim of this study was to determine if benign human uterine leiomyoma (fibroid) cells could be malignantly transformed in vitro by continuous Cd exposure and, if so, explore a molecular mechanism by which this could occur. We found when fibroid cells were exposed to 10 µM CdCl2 for 8 weeks, a robust and fast-growing Cd-Resistant Leiomyoma (CR-LM) cell culture was established. The CR-LM cells formed viable colonies in soft agar and had increased cytoplasmic glycogen aggregates, enhanced cell motility, a higher percentage of cells in G2/M phase, and increased expression of the proliferation marker Ki-67. NanoString analysis showed downregulation of genes encoding for extracellular matrix (ECM) components, such as collagens, fibronectins, laminins, and SLRP family proteins, whereas genes involved in ECM degradation (MMP1, MMP3, and MMP10) were significantly upregulated. A volcano plot showed that the top differentially genes favored cancer progression. Functional analysis by ingenuity pathway analysis predicted a significant inhibition of TGFB1 signaling, leading to enhanced proliferation and attenuated fibrosis. Prolonged Cd exposure altered phenotypic characteristics and dysregulated genes in fibroid cells predicative of progression towards a cancer phenotype. Therefore, continuous Cd exposure alters the benign characteristics of fibroid cells in vitro, and Cd exposure could possibly pose a health hazard for women with uterine fibroids.

ENVIRONMENTAL FUNGAL LOADS IN AN INDOOR-OUTDOOR AFRICAN PENGUIN ( SPHENISCUS DEMERSUS) EXHIBIT.

Aspergillosis continues to be one of the most important causes of disease in captive penguins. As such, designing exhibits and holding areas that minimize the risk of aspergillosis is of great interest; however, very little has been published regarding this topic. The goal of this study was to assess total fungal spore loads as well as the loads of Aspergillus spp. encountered in multiple indoor and outdoor microenvironments around the exhibit for a large colony of African penguins ( Spheniscus demersus). Air samples were collected via impaction at the microenvironments on a monthly basis over a 1-yr period. Results of this study indicated seasonal trends in both total fungal spore loads as well as Aspergillus spp. loads, with the lowest levels encountered during January through April. During the warmer, more humid spring, summer, and fall months when outdoor microenvironments experienced the highest fungal loads, the air-handling system and the pleated filters used indoors are thought to have reduced the fungal loads in the indoor microenvironments compared with the outdoor microenvironments. Additionally, surrounding planting beds were thought to contribute to the higher total fungal loads and Aspergillus spp. loads in the outdoor microenvironments. Results of this study are useful in understanding the factors that contribute to Aspergillus spp. loads in areas that house penguins, and can be used in guiding design, construction, and landscaping of penguin enclosures.

Fusarium solani species complex associated with carapace lesions and branchitis in captive American horseshoe crabs Limulus polyphemus.

Captive American horseshoe crabs Limulus polyphemus housed at the National Aquarium presented with a variety of shell and gill lesions over a 3 yr period. Carapace lesions were located on both the dorsal and ventral prosoma and opisthosoma and included multifocal circular areas of tan discoloration, ulcerations, and/or pitting lesions, extending from superficial to full thickness. Gill lesions involved both the book gill cover (operculum) and individual book gill leaflets and included multifocal circular areas of tan discoloration, tan to off-white opaque proliferative lesions, and/or areas of black discoloration. Histopathology revealed fungal hyphae, with variable morphology throughout the thickened and irregular cuticle of the carapace and occasionally penetrating into subcuticular tissues, with associated amebocytic inflammation. Book gill leaflets were infiltrated by fungal hyphae and contained necrotic debris and amebocytes. Thirty-eight of 39 animals (97%) evaluated via histopathological examination had intralesional fungal hyphae. Fungal cultures of carapace and gill lesions were attempted in 26 tissue samples from 15 individuals and were positive in 13 samples (50%), with 10 cultures (77%) yielding identification to genus. Fusarium sp. was identified in 8 of the 10 cultures (80%) via culture morphology. The Fusarium solani species complex was confirmed in 6 of these 8 (75%) via polymerase chain reaction amplification of 2 different ribosomal-specific sequences of isolated fungal DNA. Ante-mortem systemic and topical treatments were performed on some affected individuals, but no appreciable change in lesions was observed. Mycotic dermatitis and branchitis are serious health issues for captive American horseshoe crabs.

Capillary pressure as related to water holding in polyacrylamide and chicken protein gels.

UNLABELLED: The ability of food gels to hold water affects product yield and organoleptic quality. Most researchers believe that water is held by capillarity such that gels having smaller mean pore diameter and a more hydrophilic surface hold water more tightly. To date, however, only qualitative evidence relating pore size to water holding (WH) properties has been provided. The present study sought to provide quantitative confirmation of this hypothesis. Scanning electron microscopy coupled with image analysis was used to measure pore size, and water contact angle with the gel surface was measured by the captive bubble method, in both model polyacrylamide gels and heat-induced protein (minced chicken breast) gels. These were related to water lost during cooking of meat pastes to form gels (cooking loss (CL)), as well as water lost upon centrifugation (expressible water (EW)) or by capillary suction (CSL) of all prepared gels, as inverse measures of WH. As predicted by the Young-Laplace equation for calculating capillary pressure, the presumed mechanism of WH, gels with lower water losses exhibited a more hydrophilic surface (smaller contact angle). Yet, both lower CL and CSL correlated with larger mean pore diameter of gels, not smaller as had been expected. Polyacrylamide gels varied more in WH than did prepared meat gels, yet only the capillary suction method was sensitive enough to detect these differences. PRACTICAL APPLICATION: The ability of gels to hold water is important for economics of processing, food quality, and food safety. This study investigated the prevailing theory for how gels hold water, capillarity. Both the pore sizes of gel microstructures and the degree of hydrophilicity of the polymers comprising each gel were quantitatively assessed and related to water holding (WH) properties, and this was the first report using such methodologies. It appeared that the degree of hydrophilicity was much more important explaining WH properties than pore size, and that future research of this kind should be carried out.

Comb candidiasis affecting roosters in a broiler breeder flock.

A cutaneous mycosis caused by Candida albicans that involved the combs and less frequently the wattles, facial skin, ear lobes, and neck of male broiler breeders is described. Roosters were 35 wk old and housed with hens in two conventional broiler breeder houses on a farm in western North Carolina. Morbidity was approximately 10% in one house and less than 2% in the other house. Mortality and flock fertility were not affected. Three birds from the most affected house were examined. All birds had white adherent material on their combs that presented as crusty patches or lighter diffuse areas. Often, lesions were roughly circular or had a defined margin. Small black scabs were present in a few lesions. Similar but less extensive lesions were located on the wattles, facial skin, ear lobes, and rictus. In one bird, lesions extended down the neck, and they were accompanied by hyperemia and feather loss. Hyperkeratosis with little to no inflammation and intralesional fungi occurring as yeast and pseudohyphae were seen microscopically. High numbers of C. albicans were isolated and identified from the lesions.

Disseminated Aspergillus flavus infection in broiler breeder pullets.

Increased morbidity and mortality occurred in a 5-wk-old broiler breeder replacement pullet flock. The affected broiler pullet flock was housed on the first floor of a two-story confinement building. Mortality increased to 0.1%/day compared to the flock on the second floor, which had mortality levels of less than 0.01%/day. Clinical signs in the affected chickens included inactivity, decreased response to stimuli, and anorexia. No respiratory or neurologic signs were observed. On necropsy, affected pullets were dehydrated and emaciated and had disseminated variably sized single or multiple heterophilic granulomas that contained intralesional septate and branching fungal hyphae. Lesions were extensive around the base of the heart in the thoracic inlet and in the kidneys. Other affected organs included eyelid, muscle, proventriculus, ventriculus, intestine, liver, spleen, lung, and heart. Aspergillus flavus was cultured from the visceral granulomas. The source of flock exposure to the organism was not determined.

Acute adverse effects of the indenopyridine CDB-4022 on the ultrastructure of sertoli cells, spermatocytes, and spermatids in rat testes: comparison to the known sertoli cell toxicant Di-n-pentylphthalate (DPP).

Acute effects of CDB-4022 on testicular ultrastructure were determined. Rats were treated orally with vehicle or a maximally effective single dose of CDB-4022 or Di-n-pentylphthalate (DPP). Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. Disruption of Sertoli cell ultrastructure was apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. The severity of these degenerative effects increased at 6 and 12 hours: Vacuoles were apparent; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours, spermatocytes and spermatids were adversely affected by CDB-4022 treatment with swelling of the nuclear envelope. The Step 8 spermatids were especially noteworthy; chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete or broken, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was observed by 12 hours. CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. A rapid and direct effect of CDB-4022 on spermatocytes and spermatids was observed. The antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on Sertoli and germ cells.

Piscinoodinium, a fish-ectoparasitic dinoflagellate, is a member of the class dinophyceae, subclass gymnodiniphycidae: convergent evolution with Amyloodinium.

All dinoflagellates that infest the skin and gills of fish have traditionally been placed within the class Blastodiniphyceae. Their relatedness was primarily based upon a similar mode of attachment to the host, i.e., attachment disc with holdfasts. Results of recent molecular genetic analyses have transferred these parasites, including Amyloodinium, to the class Dinophyceae, subclass Peridiniphycidae. In our study, a small subunit rDNA gene from a parasitic dinoflagellate that has features diagnostic for species in the genus Piscinoodinium, i.e., typical trophont with attachment disc having rhizocysts, infesting the skin of freshwater tropical fish, places this organism within the dinophycean subclass Gymnodiniphycidae. This suggests a close relationship of Piscinoodinium spp. to dinoflagellates that include symbionts, e.g., species of Symbiodinium, and free-living algae, e.g., Gymnodinium spp. These molecular and morphological data suggest that evolution of this mode of fish ectoparasitism occurred independently in 2 distantly related groups of dinoflagellates, and they further suggest that the taxonomic status of parasites grouped as members of Piscinoodinium requires major revision.

Aviary air-handler design and its relationship to fungal spore loads in the air.

Fungal spore loads in the air of cool-temperature, temperate, and tropical aviaries were collected with an Andersen N-6 air sampler. The relationship of spore loads to air-handler and exhibit design in these three environments was examined. In addition, a 2-yr longitudinal study of fungal spore loads in the air of a newly designed and installed air-handling system in the R. J. Reynolds Forest Aviary at the North Carolina Zoological Park was compared to the earlier air-handling system that it replaced. High-efficiency particulate air filters installed in cool-temperature aviaries produced the cleanest air, although pleated filters showed only marginally higher spore loads. Temperate and tropical aviaries with pleated filters or bag filters with variable-velocity fans had much higher spore loads. Tropical and temperate exhibits with bag filters and constant-velocity fans produced the cleanest air in tropical and temperate exhibits. Information on the relative effectiveness of different air-handling system designs and related costs/benefits should be used by zoo managers when they are designing or retrofitting aviary air-handling systems.

Hydrocarbon synthesis by enzymatically dissociated oenocytes of the abdominal integument of the German Cockroach, Blattella germanica.

In insects, hydrocarbons waterproof the cuticle, protect the insect from the external environment, and serve as semiochemicals or their metabolic precursors. In the German cockroach, Blattella germanica, hydrocarbons are synthesized by the abdominal integument, but the precise site of biosynthesis is not known. We developed a method for separation of oenocytes from other cells in the abdominal integument using enzymatic dissociation followed by Percoll gradient centrifugation. Radiolabeled propionate was then used to monitor de novo synthesis of hydrocarbons by dissociated cells. Oenocyte-enriched cell suspensions of abdominal sternites synthesized hydrocarbons, whereas suspensions enriched with epidermal cells did not. Our results show conclusively that hydrocarbons are produced by oenocytes not only in insects whose oenocytes are localized within the hemocoel, but also in those insects whose oenocytes are within the abdominal integument. Furthermore, these data support a hemolymph pathway for transport and delivery of hydrocarbons to both external and internal tissues, including the epicuticle, fat body, and ovaries.

Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline trichomonal diarrhea.

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, a n organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Suggested Standard Operating Procedures (SOPs) for the preparation of electron microscopy samples for toxicology/pathology studies in a GLP environment.

We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentrations during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneous light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4 degrees C in McDowell's and Trump's 4F:1G fixative (4F:1G).

Pfiesteria piscicida and Ulcerative Mycosis of Atlantic Menhaden-Current Status of Understanding.

Ulcerative lesions in estuarine finfish are associated with a variety of parasitic, bacterial, and fungal infectious agents as well as water quality and other abiotic stress factors. Atlantic menhaden Brevoortia tyrannus are a severely affected species typically exhibiting solitary, perianal, focal, deep, granulomatous lesions containing oomycete hyphae. Intense recent emphasis in the scientific literature and in the popular press has been placed on Pfiesteria-like dinoflagellates and their toxins as the causative agents for ulcerative finfish lesions in east coast estuaries of the United States. Dramatic descriptive terms for Pfiesteria, including "phantom," "ambush-predator" and "the cell from hell," capable of affecting humans, have appeared in scientific journals and the news media. However, there is no scientific evidence to date that supports a single, causal relationship between the presence of toxic Pfiesteria-like dinoflagellates and fish kills or the associated ulcerative lesions. This is due, at least in part, to the difficulty of maintaining toxic Pfiesteria-like dinoflagellates in clonal culture and, in part, to the presence of other microorganisms in laboratory culture systems. Further, there is at present no toxin-specific probe to identify Pfiesteria toxins in estuarine environments during fish kills, nor has Pfiesteria been isolated from fish lesions. Yet, based on the limited published scientific evidence and the popular press coverage, there have been river closures, notable economic losses, and public alarm due to Pfiesteria. Future research into agents infectious to menhaden, such as Aphanomyces (Oomycetes) and toxin-producing estuarine inhabitants like Pfiesteria, is needed. These organisms and the disease processes in which they are implicated may also serve as markers for overall estuarine condition. Control of microorganisms, including Pfiesteria, to improve finfish health in estuaries is unlikely. However, using the presence of these microorganisms and their Atlantic menhaden hosts as sentinels to help monitor water quality may be a viable approach to assist future estuarine management.