Characterization of antimicrobial properties of Salmonella phage Felix O1 and Listeria phage A511 embedded in xanthan coatings on Poly(lactic acid) films.

Beyond simply providing a barrier between food and external contaminants, active packaging technologies aim to inhibit pathogen survival and growth within the packaged environment. Bacteriophages have a proven track record as targeted antimicrobials but have yet to be successfully integrated in active packaging without serious loss of activity. We have developed two bacteriophage based xanthan coatings on poly(lactic acid) (PLA) film which significantly inhibits Salmonella Typhimurium and Listeria monocytogenes growth in culture (P < 0.01), and significantly reduces survival and growth of diverse cocktails of Salmonella sp. and L. monocytogenes respectively on precooked sliced turkey breast over 30 days of anaerobic packaging at 4 or 10 °C (P < 0.05). Specifically reductions of 0.832 log at 4 °C and 1.30 log at 10 °C for Salmonella sp., and 6.31 log at 4 °C and 1.52 log at 10 °C for L. monocytogenes were observed. The coating containing Listeria phage A511 also significantly inhibited growth of L. monocytogenes over 14 days in aerobic packaging (3.79 log at 4 °C, 2.17 log at 10 °C, P < 0.05). These coatings showed 99.99% phage release within 30 min for both phages. Similar approaches could be used to develop packaging inhibitory to other significant foodborne pathogens such as Campylobacter, and Escherichia coli, as well as spoilage bacteria.

Effect of cranberry pomace on the physicochemical properties and inactivation of Salmonella during the manufacture of dry fermented sausages.

The effect of cranberry pomace (CP) incorporation on S. enterica serovars inactivation, starter culture population, and physicochemical properties of sausages during the manufacture of dry fermented sausages (DFS) was studied. Sausages containing a five-strain cocktail of S. enterica serovars at 7-log CFU/g, with different levels of CP (control, 0%; low, 0.55%; medium, 1.70%; high, 2.25% wt/wt), or liquid lactic acid (0.33% vol/wt, LA) were subjected to typical fermentation and drying conditions. A significant (P < 0.05) reduction in initial pH was observed in all CP treatments on day 0 as a result of CP native acidity. All treatments except low CP showed a significantly lower pH than the control throughout the study. Water activity (aw) was not significantly affected by CP level during fermentation. However, sausages containing medium and high CP levels showed a significantly lower final product aw than the control. DFS with CP exhibited a significantly (P < 0.05) faster and greater Salmonella inactivation during the first 5 days; reduction rate and level directly correlated to CP level. In the presence of medium and high levels of CP, Staphylococcus spp. growth was suppressed, while Lactobacillus spp. and Pediococcus spp. exhibited a stimulatory response. All treatments except low CP had no significant effect on product chemical composition, and Moisture Protein ratio (MPr). Low CP level yielded DFS with a slightly higher (P < 0.05) moisture content and MPr. Medium and high CP levels resulted in darker, duller and redder DFS with a softer texture. Findings suggest that low CP levels can be utilized by DFS manufacturers as a natural functional ingredient to further minimize the risk associated with Salmonella during DFS production without altering final product characteristics.

Use of 2-hydroxy-4-(methylthio)-butanoic acid to inhibit Salmonella and Listeria in raw meat for feline diets and palatability in domestic cats.

While the raw pet food market continues to grow, the risk of bacterial contamination in these types of diets is a major concern, with Salmonella enterica and Listeria monocytogenes being the most frequently associated pathogens in raw pet food product recalls. dl-Methionine is included in some commercial feline kibble and canned diets to improve protein quality; however, an alternative to this is a liquid methionine supplement, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), which is also an organic acid. 2-Hydroxy-4-(methylthio)-butanoic acid has previously demonstrated similar efficacy to formic acid against pathogens in a liquid environment and may be a good candidate to inhibit S. enterica and L. monocytogenes in raw ground meat. First, the minimum inhibitory concentration and minimum bactericidal concentration of HMTBa against these pathogens under laboratory growth conditions were determined by measuring growth of pathogens over 36 h when exposed to 10 concentrations of HMTBa (0.10% to 1.00%) mixed with tryptic soy broth. 2-Hydroxy-4-(methylthio)-butanoic acid included at ≥0.50% was bactericidal to S. enterica and L. monocytogenes (P < 0.05). Next, five levels of HMTBa (0.50% to 1.25%) were included in raw ground meat mixtures inoculated with cocktails of S. enterica or L. monocytogenes, and contamination levels were determined at four timepoints: immediately, and after refrigerated storage (4 °C) at 24, 48, and 72 h after removal from freezer (24 h at -20 °C). 2-Hydroxy-4-(methylthio)-butanoic acid included as 1.25% of the meat mixture reduced S. enterica and L. monocytogenes compared with the control (P < 0.05); however, it did not result in total kill of either of these pathogens. Following this, feeding behaviors of seven domestic cats were assessed when offered a raw chicken diet treated with or without 1.25% HMTBa for 5 d each, after which a 2-d 2-choice preference test was conducted. Cats demonstrated a preference for raw diets without HMTBa, but still readily consumed diets with 1.25% HMTBa, suggesting that such a diet was still palatable to them.

Co-operativity in agonist binding at the D2 dopamine receptor: evidence from agonist dissociation kinetics.

There is much evidence to suggest that G protein coupled receptors exist as oligomers but the relevance to their function is unclear. We have, therefore, examined the binding of the radiolabelled agonist [(3)H]NPA to membranes of CHO cells expressing the D(2) dopamine receptor in dissociation rate experiments. When [(3)H]NPA dissociation was started by dilution, the dissociation rate in the absence of sodium ions was unaffected by addition of the antagonist/inverse agonist (+)-butaclamol, but was accelerated by addition of agonists e.g. dopamine, suggesting that the receptor was not behaving as a monomer with a single binding site. The very low efficacy partial agonist, aripiprazole provided an intermediate level of acceleration of dissociation. [(3)H]NPA dissociation experiments started by addition of ligands without dilution gave a similar pattern. [(3)H]NPA dissociation could also be accelerated by GTP. Dissociation of [(3)H]NPA in the presence of GTP and dopamine provided a greater acceleration than for either modulator alone, suggesting synergistic effects related to receptor/G protein interaction. When [(3)H]NPA dissociation experiments were performed in the presence of sodium ions, dissociation was faster than in their absence but the rate still depended on the ligand present in the assay. Overall the data cannot be explained by a ternary complex model and are consistent with an oligomeric receptor in which binding of [(3)H]NPA, as an example of an agonist ligand, can be modulated co-operatively by ligands binding elsewhere in the oligomer. Interactions with G proteins also occurs providing further modulation of [(3)H]NPA binding. Both agonists and G proteins are proposed to modulate the oligomer by switching high affinity agonist binding sites to low affinity sites.

A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant.

Recent high resolution structures of modified G-protein coupled receptors (GPCRs) have provided major insight into the mechanisms of receptor-ligand binding. However understanding of the complete mechanism of GPCR function remains limited. This study characterised C-terminally truncated versions of the human adenosine A(2A) receptor (A(2A)R) with a view to producing protein suitable for structural studies. The constructs terminated at residue A316, removing the intracellular C-terminal tail, or V334, producing a C-terminal tail equivalent in length to that of rhodopsin. Higher levels of functional receptor before and after solubilisation were obtained for both C-terminally truncated constructs compared to the wild-type receptor (WT) as assessed by radioligand binding analysis using [(3)H]ZM241385. The construct which yielded the highest level of functional receptor, V334 A(2A)R, was purified in DDM to high homogeneity with a final yield of 2 mg/L. Binding analysis revealed that the purified receptor had a specific activity of 20.2+/-1.2 nmol/mg, close to the theoretical maximum. Pure V334 A(2A)R was resistant to degradation over 15 days when stored at 4 degrees C or 20 degrees C and showed remarkable functional stability when stored at 4 degrees C, retaining 84% of initial functionality after 30 days. This construct is an excellent candidate for structural studies.

Antipsychotic drug action: antagonism, inverse agonism or partial agonism.

The positive, psychotic symptoms of schizophrenia can be treated by antipsychotic drugs and it has been assumed that these are antagonists at the D(2) and D(3) dopamine receptors in the brain. Recently, the D(2)/D(3) partial agonist aripiprazole has been introduced as an antipsychotic drug. It has also been realized that, using in vitro assays, the other antipsychotic drugs are in fact inverse agonists at D(2)/D(3) dopamine receptors. This raises questions about how these disparate drugs can achieve a similar clinical outcome. In this review, I shall consider the efficacies of these drugs in signalling assays and how these efficacies might affect treatment outcomes. It seems that the treatment outcome might depend on the overall level of cell stimulation, which is in turn dependent on the level of residual dopamine and the efficacy of the drug in signalling assays.

Signaling mechanisms of GPCR ligands.

G protein-coupled receptors constitute one of the major classes of drug targets, so understanding the mechanisms of signaling through these receptors is of great importance. This review covers some of the recent advances in G protein-coupled receptor signaling. A high resolution structure of the beta 2-adrenergic receptor has been reported, as well as several molecular switches involved in receptor activation. It has also been realised that receptors and G proteins and their subunits may not always separate upon receptor activation. The definition of the ability of these receptors to signal has been expanded considerably with the realisation that some signaling may occur independently of G proteins, that some signaling events may differ in their pharmacological profiles and that formation of heterodimers of these receptors may provide new avenues for both signaling and drug design.

Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures.

BACKGROUND: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. RESULTS: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies. CONCLUSION: Large-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

Effects of High Pressure Processing and Hot Water Pasteurization of Cooked Sausages on Inactivation of Inoculated Listeria monocytogenes, Natural Populations of Lactic Acid Bacteria, Pseudomonas spp., and Coliforms and Their Recovery during Storage at 4 and 10°C.

The study investigated the effects of high pressure processing (HPP; 600 MPa for 3 min) and hot water (HW; 75°C for 15 min) pasteurization on the inactivation of inoculated Listeria monocytogenes, natural populations of lactic acid bacteria, Pseudomonas spp., and coliforms in vacuum-packaged cooked sausages and their recovery during storage at 4 and 10°C for 35 days. Cooking sausages to an internal temperature of 72°C resulted in a >6-log reduction in numbers of inoculated L. monocytogenes. Storage at 4°C resulted in no significant difference ( P > 0.05) in L. monocytogenes numbers in sausages pasteurized by either HPP or HW compared with unpasteurized control. However, at 10°C, L. monocytogenes numbers in unpasteurized control sausages increased to about 7 log CFU/g by day 35, whereas in HPP-pasteurized sausages, numbers remained below the detection limit for up to 21 days and then increased to 4.5 log CFU/g by day 35. HW pasteurization resulted in inhibition of L monocytogenes to below the detection limit throughout the 35-day storage at 10°C. Natural lactic acid bacteria populations were significantly reduced by HPP and HW pasteurization and continued to be significantly lower at the end of the 35-day storage. Unlike most studies that focus on HPP or HW treatment of postcooking surface contamination of meat with Listeria, this study examined the combined effect of cooking, HPP, and HW on raw meat with a high contamination level. This scenario is important in countries where raw meat supply and in-store refrigeration are a challenge. The results suggest that HPP and HW pasteurization could be used to successfully enhance the safety and shelf life of cooked sausages and that HW pasteurization (75°C) was more effective than HPP (600 MPa) to control L. monocytogenes.

Effect of caliber size and fat level on the inactivation of E. coli O157:H7 in dry fermented sausages.

Dry fermented sausages (DFS) have been subject to numerous validation studies, as pathogen reduction heavily relies on both ingredients and processing. In this study the effect of product caliber size (32, 55, 80mm), and fat level (low, 9.67%; high, 18.46% wt/wt) on the inactivation of E. coli O157:H7 during DFS production was examined. Sausages containing a five-strain cocktail of E. coli O157:H7 at 107CFU/g were manufactured and monitored for changes in physicochemical properties and inoculated E. coli O157:H7 numbers were enumerated during the DFS production stages and log reduction rates were calculated. Significant (P<0.01) reduction in pH from 5.9 to 4.9 was observed in all sausages within 72h of fermentation; however, the observed pH reduction was not significantly (P>0.05) different among sausages of different caliber size or fat levels. No significant (P>0.05) reduction in aw was observed during fermentation of the sausages. However, during the drying phase, sausages with larger caliber sizes required a significantly longer duration of drying to achieve the same aw of smaller caliber size sausages. For instance, to achieve an aw of ≤0.9, following 5days of fermentation/curing, 80mm caliber sausages required up to 27days of drying compared with 13 and 6days for 55 and 32mm caliber size sausages, respectively. Fat levels on the other hand did not significantly (P>0.05) effect the reduction of aw during drying of the sausages. During the fermentation stage there was a significant and rapid reduction in E. coli O157:H7 counts by about 1.1- to 1.4-log units, but was not significantly different among sausages of different caliber size and fat levels. Considering the whole process, only caliber size had a significant effect on log reduction of E. coli O157:H7. ANOVA of log reduction rates of E. coli O157:H7 among sausages of different caliber size and fat levels revealed no significant differences during the fermentation, however, during the drying of the sausages, log reduction rate of E. coli O157:H7 was significantly (P<0.01) lower in sausages with larger caliber sizes and higher fat levels. For instance, log reduction rates for E. coli O157:H7 in high fat large caliber sausages was the lowest at -0.082±0.004 log CFU/g/day compared to all other fat and caliber size combinations. These results suggest that DFS manufacturers producing higher fat and large caliber size products need to consider longer drying periods to achieve the required 5-log inactivation of E. coli O157:H7.

Genomic and Proteomic Analyses of Salmonella enterica Serovar Enteritidis Identifying Mechanisms of Induced de novo Tolerance to Ceftiofur.

With the alarming proliferation of antibiotic resistance, it is important to understand the de novo development of bacterial adaptation to antibiotics in formerly susceptible lineages, in the absence of external genetic input from existing resistance pools. A strain of ceftiofur susceptible Salmonella enterica serovar Enteritidis ABB07-SB3071 (MIC = 1.0 µg/ml) was successively exposed to sub-MIC of ceftiofur to allow its adaptation for tolerance to a concentration of 2.0 µg/ml of this antibiotic. Genomic and proteomic comparative analyses of the parental strain and induced tolerant derived lineages were performed to characterize underlying mechanisms of de novo adaptation (tolerance). Expression and localization of specific drug-, heme-, sugar-, amino acid-, and sulfate-transporters were altered, as was the localization of the cell membrane stabilizing protein OsmY in the tolerant strains adapted to 2.0 µg/ml compared to the parental isolate lines. This redistribution of existing transporters acts to minimize the concentrations of ceftiofur in the periplasm, by decreasing facilitated import and increasing active efflux and cytosolic sequestration as determined by high performance liquid chromatography quantification of residual total and extracellular ceftiofur after growth. Genetic, subcellular localization, and abundance changes of specific regulators of transcription, translation, and post-translational dynamics in the derived ceftiofur tolerant lineages decrease metabolic strain on cell walls and enhance periplasmic envelop stability against stress. This produces slower growing, more tolerant populations, which deplete free ceftiofur concentrations significantly more than susceptible parental populations (P < 0.05), as measured by recoverable levels of ceftiofur from cultures of equivalent cellular density incubated with equal ceftiofur concentrations. Genetic and abundance changes to specific carbon and nitrogen metabolism enzymes, not traditionally associated with beta-lactam metabolism, establish an enzymatic framework with the potential to detoxify/degrade ceftiofur, while mutations and changes in subcellular localization in specific cell surface factors enhance the stability of the Gram-negative cell envelop despite the compromising effect of ceftiofur. The observed changes highlight generalizable mechanisms of de novo tolerance without horizontal gene transfer, and thus can inform policies to combat antibiotic tolerance and minimize induction of de novo tolerance.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5.

Antagonists of the chemokine receptor, CCR5, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCR5 (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [(35)S]GTPgammaS binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2)-stimulated [(35)S]GTPgammaS binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (approximately 15%) in cell surface CCR5 was detected.

Diverse signalling by different chemokines through the chemokine receptor CCR5.

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

Examining the Effects of Sodium Ions on the Binding of Antagonists to Dopamine D2 and D3 Receptors.

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo.

Mechanisms of inverse agonism at G-protein-coupled receptors.

Many drugs with important therapeutic actions that had been assumed to be antagonists at G-protein-coupled receptors (GPCRs) have been shown to be inverse agonists. For both basic pharmacology and drug design it is important to understand the mechanisms whereby these drugs achieve their effects. It had been assumed that these drugs achieved their effects by stabilizing an inactive state of the receptor (R) at the expense of a partially activated state (R*). In this article, I consider this and other mechanisms that could explain inverse agonist actions, and conclude that more than one mechanism can apply to inverse agonism at GPCRs.

Mechanisms of inverse agonist action at D2 dopamine receptors.

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K(i) values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K(coupled) and K(uncoupled) were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTP gamma S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Oligomers of D2 dopamine receptors: evidence from ligand binding.

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D2 dopamine receptor and have shown using a variety of biochemical and biophysical techniques that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands.

An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products.

Finger millet is an ancient African cereal crop, domesticated 7000 years ago in Ethiopia, reaching India at 3000 BC. Finger millet is reported to be resistant to various fungal pathogens including Fusarium sp. We hypothesized that finger millet may host beneficial endophytes (plant-colonizing microbes) that contribute to the antifungal activity. Here we report the first isolation of endophyte(s) from finger millet. Five distinct fungal species were isolated from roots and predicted taxonomically based on 18S rDNA sequencing. Extracts from three putative endophytes inhibited growth of F. graminearum and three other pathogenic Fusarium species. The most potent anti-Fusarium strain (WF4, predicted to be a Phoma sp.) was confirmed to behave as an endophyte using pathogenicity and confocal microscopy experiments. Bioassay-guided fractionation of the WF4 extract identified four anti-fungal compounds, viridicatol, tenuazonic acid, alternariol, and alternariol monomethyl ether. All the purified compounds caused dramatic breakage of F. graminearum hyphae in vitro. These compounds have not previously been reported to have anti-Fusarium activity. None of the compounds, except for tenuazonic acid, have previously been reported to be produced by Phoma. We conclude that the ancient, disease-tolerant crop, finger millet, is a novel source of endophytic anti-fungal natural products. This paper suggests the value of the crops grown by subsistence farmers as sources of endophytes and their natural products. Application of these natural chemicals to solve real world problems will require further validation.