Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH).

Aerobic plate counts, the standard for bacterial enumeration in the probiotic industry, can be biased towards fast-growing organisms that replicate on synthetic media and can significantly underestimate total bacterial abundance. Culture-independent approaches such as fluorescence in situ hybridization (FISH) hold promise as a means to rapidly and accurately enumerate bacteria in probiotic products. In addition, FISH has the potential to more accurately represent bacterial growth dynamics in the environment in which products are applied without imposing additional growth constraints that are required for enumeration via plate counts. In this study, we designed and optimized three new FISH probes to visualize and quantify Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus licheniformis within probiotic products. Microscopy-based estimates were consistent or higher than label claims for Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus in both a direct fed microbial (DFM) product as well as a crop microbial biostimulant (CMB) product. Quantification with FISH after a germination experiment revealed the potential for this approach to be used after application of the product.

3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

Novel HTS strategy identifies TRAIL-sensitizing compounds acting specifically through the caspase-8 apoptotic axis.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS) strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be "weeded out" in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC) was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries.

The phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures.

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in many human tumors and mediates processes such as cell proliferation, survival, adhesion, and motility. The natural product, wortmannin, has been widely used to study the functional consequences of PI3K inhibition in both normal and transformed cells in culture but is not a suitable cancer chemotherapeutic agent due to stability and toxicity issues. PX-866, an improved wortmannin analogue, displays significant antitumor activity in xenograft models. Here, we directly compare PX-866 and wortmannin in human cancer cell lines cultured in monolayer or as three-dimensional spheroids. Both PI3K inhibitors failed to inhibit monolayer cell growth at concentrations up to 100 nmol/L but strongly suppressed spheroid growth at low nanomolar concentrations, with PX-866 showing greater potency than wortmannin. Relative to wortmannin, PX-866 treatment results in a more sustained loss of Akt phosphorylation, suggesting that the increased potency of PX-866 is related to a more durable inhibition of PI3K signaling. PX-866 and wortmannin both inhibit spheroid growth without causing cytotoxicity, similar to known cytostatic agents, such as rapamycin. PX-866 also inhibits cancer cell motility at subnanomolar concentrations. These findings suggest that the antitumor activities of PX-866 stem from prolonged inhibition of the PI3K pathway and inhibition of cell motility. In addition, we propose that the use of three-dimensional tumor models is more predictive of in vivo growth inhibition by PI3K inhibitors in cancer cell lines lacking phosphatase and tensin homologue activity or expression.

Galphaq expression activates EGFR and induces Akt mediated cardiomyocyte survival: dissociation from Galphaq mediated hypertrophy.

Our laboratory has previously shown that adenoviral-mediated overexpression of Galphaq in neonatal rat ventricular cardiomyocytes increases the phosphorylation of Akt, a well-established anti-apoptotic effector. As demonstrated here, Galphaq expression protects cardiomyocytes against apoptosis induced by treatment with 2-deoxyglucose (2DOG) and this protection is lost when Akt activation is prevented by treatment with LY294002 (an inhibitor of PI3K). Galphaq-induced Akt phosphorylation is not caused by increased Gbetagamma signaling and does not appear to involve PKC activation. Rather studies using the EGF receptor inhibitor AG1478 and the Src inhibitor PP2 implicate these tyrosine kinases in the pathway inducing Akt phosphorylation. EGFR phosphorylation is increased in cells expressing Galphaq and this effect is inhibited by PP2, placing Src upstream of EGFR phosphorylation. EGFR activation appears to be required for Galphaq-mediated protection since inhibition of Src or EGFR rendered cells susceptible to 2DOG-induced apoptosis. In contrast to the requirement for EGFR mediated Akt activation in cardioprotection, neither EGFR nor Akt activation are necessary for the hypertrophic increases in cell size or ANF content elicited by Galphaq overexpression. These data demonstrate that increased Galphaq activity can provide anti-apoptotic signals by eliciting EGFR phosphorylation and subsequent Akt activation, independent of the well-known ability of Galphaq signaling to elicit hypertrophy.

Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT.

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.

Upregulation of GLUT1 expression is necessary for hypertrophy and survival of neonatal rat cardiomyocytes.

During hypertrophy the heart increases its utilization of glucose and decreases that of fatty acids, resuming a fetal pattern of substrate metabolism. As demonstrated here, GLUT1 protein expression is increased in association with in vivo pressure-overload-induced hypertrophy. The relationship of changes in GLUT1 to enhanced glucose uptake and to cardiomyocyte hypertrophy and survival is not known. To explore this question we first examined the effect of prostaglandin F2alpha (PGF2alpha), an established hypertrophic agonist, on GLUT1 expression and glucose uptake in neonatal rat ventricular myocytes (NRVMs). PGF2alpha treatment for 24 h led to a fivefold increase in GLUT1 expression and a sixfold increase in glucose uptake. However, NRVMs cultured in the absence of glucose or with 3-O-methyl glucose, a competitive inhibitor of glucose uptake, still exhibited PGF2alpha-induced hypertrophic growth. In addition, we determined that overexpression of GLUT1 using adenovirus was insufficient to cause an increase in cell size, myofibrillar organization, or atrial natriuretic factor (ANF) expression. On the other hand, adenoviral overexpression of antisense GLUT1 (which blocked PGF2alpha-induced increases in GLUT1 protein) prevented PGF2alpha-stimulated cell enlargement and increases in ANF transcription. Overexpression of GLUT1 or addition of PGF2alpha also protected cells against serum deprivation-induced apoptosis; this effect was blocked by antisense GLUT1 but, surprisingly, was not dependent on glucose. Together, these data suggest that upregulation of GLUT1 serves a role in agonist-induced hypertrophy and survival which can be dissociated from its role in glucose transport.

Protein kinase C epsilon-dependent activation of proline-rich tyrosine kinase 2 in neonatal rat ventricular myocytes.

Proline-rich tyrosine kinase 2 (PYK2) is a nonreceptor protein tyrosine kinase that links G-protein-coupled receptors to activation of MAPK cascades and cellular growth. In smooth muscle and other cell types, PYK2 activation is dependent on either Ca(2+) or protein kinase C (PKC), and we have previously shown that endothelin-1 (ET) activates PYK2 in adult and neonatal rat ventricular myocytes (NRVM). However, ET both alters intracellular Ca(2+) ([Ca(2+)](i)), and activates the novel, Ca(2+)-independent PKCs. Therefore, immunoprecipitation and western blotting experiments were used to examine the PKC and Ca(2+) dependence of PYK2 activation in NRVM. PYK2 was activated by ET (100 nM; 2-30 min) and phenylephrine (50 microM; 2-30 min), which are both hypertrophic agonists that activate Gq-coupled receptors. Moreover, adenoviral (Adv)-mediated overexpression of constitutively active (ca) Galphaq increased PYK2-Y(402) phosphorylation as early as 8 h post-infection, as compared to NRVM infected with a control Adv encoding beta-galactosidase. caGalphaq overexpression also induced PKC epsilon and PKCdelta (but not PKCalpha) translocation, followed by downregulation of both novel PKC isoenzymes. Phorbol myristate acetate (PMA; 200 nM), a direct activator of Ca(2+)-dependent and Ca(2+)-independent PKCs, activated PYK2 within 10 min, and PYK2 phosphorylation remained elevated after 30 min of stimulation. Adv-mediated overexpression of caPKC epsilon increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKC epsilon markedly inhibited ET-induced, but not basal PYK2 phosphorylation. In contrast, both basal and ET-induced PYK2 phosphorylation were blocked by treatment with the Src-family protein kinase inhibitor PP2. Although reducing [Ca(2+)](i) with either nifedipine (10 microM) or BAPTA-AM (50 microM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing [Ca(2+)](i) with ionomycin (10 microM), K(+) depolarization, or BayK8644 (1 microM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKC epsilon, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes.

Akt-mediated cardiomyocyte survival pathways are compromised by G alpha q-induced phosphoinositide 4,5-bisphosphate depletion.

Expression of the wild type alpha subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active G alpha q subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac G alpha q overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity.