Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.     

Impact of variance components on reliability of absolute quantification using digital PCR

Background

Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.

Results

We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.

Conclusions

Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-283) contains supplementary material, which is available to authorized users.     

TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca²⁺-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca²⁺ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca²⁺-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca²⁺-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca²⁺ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca²⁺ transients partially rely on Ca²⁺ influx in PACs, the role of TRPM4 was also assessed on Ca²⁺ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca²⁺ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca²⁺. In conclusion, TRPM4 links intracellular Ca²⁺ signaling to membrane potential as a negative feedback regulator of Ca²⁺ entry in PACs.     

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

Adjustment of juvenile tuatara to a cooler,southern climate: operative temperatures,emergence behaviour and growth rate

Translocations for conservation often involve species limited to relict distributions. However, uncertainty can exist regarding the ability of source individuals to acclimatise following a shift to a distant location. We investigated the ability of captive-reared juvenile tuatara (Sphenodon punctatus) of Cook Strait stock (41°S) to adjust to outdoor, predator-protected pens within Orokonui Ecosanctuary (45 °S). We examined potential basking and within burrow temperatures, the influence of temperature on emergence, and growth rates in comparison with other locations. Tuatara at Orokonui reached their preferred temperature when basking over summer, and burrows provided protection from freezing over winter. Emergence was temperature-dependent and essentially ceased during winter. Growth rates of Orokonui-held juveniles were within the range for four other captive-rearing facilities and faster than for wild juveniles from a Cook Strait population. As all Orokonui-held juveniles have survived and grown we conclude that the climate at this southern location is suitable to consider a free-release.     

TRPM3 is a nociceptor channel involved in the detection of noxious heat

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in?vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.     

Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16

Cytokines and other paracrine or autocrine factors functionally modulate the invasion-suppressor and signal-transducing E-cadherin/catenin complex. We have used conditioned medium from human squamous carcinoma COLO 16 cells (CM COLO 16) as a source of such factors to modulate the E-cadherin/catenin complex in human breast carcinoma MCF-7 cells. CM COLO 16 induces scattering of MCF-7/AZ, but not of MCF-7/6 cells on tissue culture plastic substratum, and reduces aggregation of MCF-7/AZ cells in suspension. Insulin-like growth factor I counteracts this reduction of aggregation. Confocal laser scanning microscopy of immunocytochemical stainings shows loss of the honeycomb pattern of E-cadherin, alpha-catenin and beta-catenin, and internalization of those elements. Cell surface biotinylation shows a decrease in membrane-bound E-cadherin. Immunoprecipitation and cell fractionation show that the composition of the complex is maintained. Interleukin-1, interleukin-6, granulocyte-monocyte colony stimulating factor, stem cell factor, scatter factor/hepatocyte growth factor and transforming growth factor-beta, added separately to MCF-7/AZ cells, could not mimic the effects of CM COLO 16. Neither could we find evidence that the 80 kDa extracellular fragment of E-cadherin is implicated in scattering of MCF-7/AZ cells. This fragment is present in CM COLO 16, but it is also produced by the MCF-7/AZ cells themselves, even at higher levels. Our data point toward cytoplasmic internalization induced by paracrine factors as one of the downregulating mechanisms for the E-cadherin/catenin complex.     

Down-regulation of the ATP-binding cassette transporter 2 (Abca2) reduces amyloid-β production by altering Nicastrin maturation and intracellular localization

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-β (Aβ) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aβ generation, without affecting Notch cleavage.