Growth and Aflatoxin B1, B2, G1, and G2 Production by Aspergillus flavus and Aspergillus parasiticus on Ground Flax Seeds (Linum usitatissimum).

ABSTRACT: Flax seed has become an increasingly popular food ingredient because of its nutrient richness as well as potential health benefits. Flax seeds are often ground before consumption, and flax seed cakes are used as animal feed. Aflatoxin production may occur subsequently when the ground seeds are stored in an environment that supports fungal growth. The objectives of this study were to determine the growth of two toxigenic fungi, Aspergillus flavus and A. parasiticus, and to quantify the concentrations of four major aflatoxins (AFB1, AFG1, AGB2, and AFG2) produced by the two fungi on ground flax seeds with water activities (aws) of 0.82, 0.86, 0.90, 0.94, and 0.98, incubated for 30 days at 20, 27, and 35°C. Results of the study showed that A. flavus was able to grow on ground seeds with aw from 0.86 to 0.98 at all three temperatures, and the most rapid growth occurred at aws 0.90 and 0.94 at 27°C. In comparison, A. parasiticus grew on seeds with aw from 0.86 to 0.98 at 27 and 35°C as well as on seeds with aw from 0.86 to 0.90 at 20°C, and the most favorable growth condition was aw from 0.90 to 0.94 at 35°C. A. flavus produced aflatoxins on seeds with aw from 0.90 to 0.94 at 27°C as well as on seeds with aw from 0.86 to 0.98 at 35°C, and the maximum total aflatoxin (298 µg/kg), AFB1 (247 µg/kg), and AFG1 (51 µg/kg) were found on seeds with aw 0.90 at 35°C. In comparison, A. parasiticus produced aflatoxins under a wider range of conditions, which included aw 0.86 at 27 and 35°C, aw 0.90 at 20 and 27°C, aw 0.94 at 27°C, and aw 0.98 at 35°C. The maximum total aflatoxin (364 µg/kg) and maximum AFB1 (324 µg/kg) along with 34 µg/kg AFG1 and 6 µg/kg AFB2 were produced by A. parasiticus on seeds with aw 0.98 incubated at 35°C for 30 days. Linear regression models also indicated that high incubation temperature (35°C) was optimal for overall fungal growth and for formation of high levels of aflatoxin by both fungi. Future studies should also address aflatoxin contamination in flax seed oil.

Aflatoxin B1 (AFB1) production by Aspergillus flavus and Aspergillus parasiticus on ground Nyjer seeds: The effect of water activity and temperature.

Nyjer oil seed cake supports high levels of aflatoxin B1 (AFB1) production. AFB1 is a secondary metabolite of Aspergillus flavus and A. parasiticus, classified as a Class 1A carcinogen. The aim of this study was to determine the effects of temperature (20, 27, and 35 °C) and water activity (0.82, 0.86, 0.90, 0.94, and 0.98 aw) on fungal growth and AFB1 production of A. flavus and A. parasiticus on ground Nyjer seeds over a 30-day incubation period. Linear regression models indicated that both fungal growth and AFB1 production were significantly influenced by water activity of Nyjer seeds and incubation temperature. The two fungi did not grow on Nyjer seeds at 0.82 aw at the three incubation temperatures. The most favorable growth conditions for both fungi were 0.90-0.98 aw at 27 °C or 0.90-0.94 aw at 35 °C. The optimum temperature for AFB1 production was 27 °C for both A. flavus and A. parasiticus (with regression coefficients of 6.01 and 9.11, respectively). Both fungi were likely to produce high levels of AFB1 at 0.90 aw (with regression coefficients of 3.56 for A. flavus and 7.17 for A. parasiticus). Aspergillus flavus only produced AFB1 on seeds with 0.90-0.98 aw at 27 °C (in the range of 203-282 µg/kg) and on seeds with 0.90 aw at 35 °C (212 µg/kg). No detectable AFB1 was produced by this fungus in any other culture conditions that were studied. Aspergillus parasiticus, in contrast, was able to produce AFB1 under all of the growth conditions. At 20 °C, this fungus produced the highest level of AFB1 (212 µg/kg) at high water activity (0.98 aw). At 27 °C, A. parasiticus produced high levels of AFB1 (in the range of 209-265 µg/kg) at a wide range of water activities (0.86-0.98 aw). In the entire study, the highest AFB1 concertation for A. parasiticus was detected on seeds incubated at high temperature (35 °C) and low water activity (0.86 aw). The findings of this study could help optimize the storage conditions of Nyjer oil seeds to reduce aflatoxin contamination.

Effect of water activity, temperature, and incubation period on fungal growth and ochratoxin A production on Nyjer seeds.

Aspergillus fresenii and Aspergillus sulphureus produce ochratoxin A (OTA), which is a secondary metabolite of Aspergillus and Penicillium species, with nephrotoxic effects and potential carcinogenic activity. The aim of this study was to determine the effects of temperature (20, 30, and 37 °C), water activity (0.82, 0.86, 0.90, 0.94, and 0.98 aw), incubation period (5, 10, 15, and 30 days) on fungal growth, and OTA production by A. fresenii and A. sulphureus on Nyjer oil seeds. There was no fungal growth at 0.82 aw. The two fungal species were able to produce OTA from the fifth day of incubation from 0.86 to 0.98 aw and temperature 20 to 37 °C. Aspergillus fresenii produced the highest concentration of OTA (643 µg/kg) at 0.90 aw and 37 °C within 15 days, while A. sulphureus produced the highest level of OTA (724 µg/kg) at 0.98 aw and 20 °C within 10 days. The optimum water activity and temperature for the growth of both fungi were similar at 0.94 aw and at 30 °C. There was statistically significant difference between the levels of OTA production among the two fungi. Overall, A. sulphureus produced significantly higher levels of OTA (p < 0.05). Higher temperature (37 °C) and 0.90-0.94 aw were optimal for OTA production by A. fresenii. Our results show that Nyjer seeds can support the growth of A. fresenii and A. sulphureus and OTA production, and the two species had similar temperature and water activity requirements for growth but different requirements for OTA production on Nyjer seeds.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease.

BACKGROUND: Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. METHODS: To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. RESULTS: We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). CONCLUSIONS: Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

Transferred NOESY NMR studies of biotin mimetic peptide (FSHPQNT) bound to streptavidin: a structural model for studies of peptide-protein interactions.

Protein-protein interactions control signaling, specific adhesion, and many other biological functions. The three-dimensional structures of the interfaces and bound ligand can be approached with transferred nuclear Overhauser effect spectroscopy NMR, which can be applied to much larger proteins than conventional NMR and requires less concentrated protein. However, it is not clear how accurately the structures of protein-bound peptides can be determined by transferred nuclear Overhauser effect spectroscopy. We studied the structure of a biotin mimetic peptide (FSHPQNT) bound to streptavidin, because the X-ray structure of the complex is available to 1.74 Å resolution, and we found that conditions could be adjusted so that the off-rates were fast enough for transferred nuclear Overhauser effect spectroscopy NMR. The off-rate was determined with (19)F NMR, using a para-fluoro-phenylalanine analog of the peptide. A new criterion for a lower limit on kinetic off-rate was found, which allowed accurate structure determination at a slower off-rate. Non-specific binding of the peptide to streptavidin was not significant, because biotin blocked the peptide transferred nuclear Overhauser effect spectroscopy. Protein mediation for the long-range peptide transferred nuclear Overhauser effect spectroscopy cross-peaks was corrected by a transferred nuclear Overhauser effect spectroscopy/ROESY averaging procedure. The protein-bound structure of the peptide was determined by transferred nuclear Overhauser effect spectroscopy constrained and simulated annealing. The structure deduced from the NMR was close to the X-ray structure.

NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6).

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.