Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

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.      

Effect of graded fructose coingestion with maltodextrin on exogenous 14C-fructose and 13C-glucose oxidation efficiency and high-intensity cycling performance.

The ingestion of solutions containing carbohydrates with different intestinal transport mechanisms (e.g., fructose and glucose) produce greater carbohydrate and water absorption compared with single-carbohydrate solutions. However, the fructose-ingestion rate that results in the most efficient use of exogenous carbohydrate when glucose is ingested below absorption-oxidation saturation rates is unknown. Ten cyclists rode 2 h at 50% of peak power then performed 10 maximal sprints while ingesting solutions containing (13)C-maltodextrin at 0.6 g/min combined with (14)C-fructose at 0.0 (No-Fructose), 0.3 (Low-Fructose), 0.5 (Medium-Fructose), or 0.7 (High-Fructose) g/min, giving fructose:maltodextrin ratios of 0.5, 0. 8, and 1.2. Mean (percent coefficient of variation) exogenous-fructose oxidation rates during the 2-h rides were 0.18 (19), 0.27 (27), 0.36 (27) g/min in Low-Fructose, Medium-Fructose, and High-Fructose, respectively, with oxidation efficiencies (=oxidation/ingestion rate) of 62-52%. Exogenous-glucose oxidation was highest in Medium-Fructose at 0.57 (28) g/min (98% efficiency) compared with 0.54 (28), 0.48 (29), and 0.49 (19) in Low-Fructose, High-Fructose, No-Fructose, respectively; relative to No-Fructose, only the substantial 16% increase (95% confidence limits +/-16%) in Medium-Fructose was clear. Total exogenous-carbohydrate oxidation was highest in Medium-Fructose at 0.84 (26) g/min. Although the effect of fructose quantity on overall sprint power was unclear, the metabolic responses were associated with lower perceptions of muscle tiredness and physical exertion, and attenuated fatigue (power slope) in the Medium-Fructose and High-Fructose conditions. With the present solutions, low-medium fructose-ingestion rates produced the most efficient use of exogenous carbohydrate, but fatigue and the perception of exercise stress and nausea are reduced with moderate-high fructose doses.     

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.     

Contribution of Lactococcus lactis Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of α_S1-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.     

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers.Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH.In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.     

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.