Observations below multiple lower limits of quantification: How to estimate the mean and variance

Multiple lower limits of quantification (MLOQs) result if various laboratories are involved in the analysis of concentration data and some observations are too low to be quantified. For normally distributed data under MLOQs there exists only the multiple regression method of Helsel to estimate the mean and variance. We propose a simple imputation method and two new maximum likelihood estimation methods: the multiple truncated sample method and the multiple censored sample method. A simulation study is conducted to compare the performances of the newly introduced methods to Helsel's via the criteria root mean squared error (RMSE) and bias of the parameter estimates. Two and four lower limits of quantification (LLOQs), various amounts of unquantifiable observations and two sample sizes are studied. Furthermore, the robustness is investigated under model misspecification. The methods perform with decreasing accuracy for increasing rates of unquantified observations. Increasing sample sizes lead to smaller bias. There is almost no change in the performance between two and four LLOQs. The magnitude of the variance impairs the performance of all methods. For a smaller variance, the multiple censored sample method leads to superior estimates regarding the RMSE and bias, whereas Helsel's method is superior regarding the bias for a larger variance. Under model misspecification, Helsel's method was inferior to the other methods. Estimating the mean, the multiple censored sample method performed better, whereas the multiple truncated sample method performs best in estimating the variance. Summarizing, for a large sample size and normally distributed data we recommend to use Helsel's method. Otherwise, the multiple censored sample method should be used to obtain estimates of the mean and variance of data including MLOQs.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Distinct molecular features of colorectal cancer in Ghana

Objectives: While colorectal cancer (CRC) is common, its incidence significantly varies around the globe. The incidence of CRC in West Africa is relatively low, but it has a distinctive clinical pattern and its molecular characteristics have not been studied. This study is one of the first attempts to analyze molecular, genetic, and pathological characteristics of colorectal cancer in Ghana. Methods: DNA was extracted from microdissected tumor and adjacent normal tissue of 90 paraffin blocks of CRC cases (1997–2007) collected at the University of Ghana. Microsatellite instability (MSI) was determined using fragment analysis of ten microsatellite markers. We analyzed expression of mismatch repair (MMR) proteins by immunohistochemistry and sequenced exons 2 and 3 of KRAS and exon 15 of BRAF. Results: MSI analysis showed 41% (29/70) MSI-High, 20% (14/70) MSI-Low, and 39% (27/70) microsatellite-stable (MSS) tumors. Sequencing of KRAS exons 2 and 3 identified activating mutations in 32% (24/75) of tumors, and sequencing of BRAF exon 15, the location of the common activating mutation (V600), did not show mutations at codons 599 and 600 in 88 tumors. Conclusions: Our study found a high frequency of MSI-High colorectal tumors (41%) in Ghana. While the frequency of KRAS mutations is comparable with other populations, absence of BRAF mutations is intriguing and would require further analysis of the molecular epidemiology of CRC in West Africa.     

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

Promiscuous mating produces offspring with higher lifetime fitness

In many species, each female pairs with a single male for the purpose of rearing offspring, but may also engage in extra-pair copulations. Despite the prevalence of such promiscuity, whether and how multiple mating benefits females remains an open question. Multiple mating is typically thought to be favoured primarily through indirect benefits (i.e. heritable effects on the fitness of offspring). This prediction has been repeatedly tested in a variety of species, but the evidence has been equivocal, perhaps because such studies have focused on pre-reproductive survival rather than lifetime fitness of offspring. Here, we show that in a songbird, the dark-eyed junco (Junco hyemalis), both male and female offspring produced by extra-pair fertilizations have higher lifetime reproductive success than do offspring sired within the social pair. Furthermore, adult male offspring sired via extra-pair matings are more likely to sire extra-pair offspring (EPO) themselves, suggesting that fitness benefits to males accrue primarily through enhanced mating success. By contrast, female EPO benefited primarily through enhanced fecundity. Our results provide strong support for the hypothesis that the evolution of extra-pair mating by females is favoured by indirect benefits and shows that such benefits accrue much later in the offspring's life than previously documented.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Synthesis and AcidBase Properties of an Imidazole-Containing Nucleotide Analog, 1-(2'-Deoxy-β-D-ribofuranosyl)imidazole 5'-Monophosphate (dImMP(2-) )

Deletion of the substituted pyrimidine ring in purine-2'-deoxynucleoside 5'-monophosphates leads to the artificial nucleotide analog dImMP(2-) . This analog can be incorporated into DNA to yield, upon addition of Ag(+) ions, a molecular wire. Here, we measured the acidity constants of H(2) (dImMP)(±) having one proton at N(3) and one at the PO$\rm{{_{3}^{2-}}}$ group by potentiometric pH titrations in aqueous solution. The micro acidity constants show that N(3) is somewhat more basic than PO$\rm{{_{3}^{2-}}}$ and, consequently, the (H??dImMP)(-) tautomer with the proton at N(3) dominates to ca. 75%. The calculated micro acidity constants are confirmed by (31) P- and (1) H-NMR chemical shifts. The assembled data allow many quantitative comparisons, e.g., the N(3)-protonated and thus positively charged imidazole residue facilitates deprotonation of the P(O)(2) (OH)(-) group by 0.3?pK units. Information on the intrinsic site basicities also allows predictions about metal-ion binding; e.g., Mg(2+) and Mn(2+) will primarily coordinate to the phosphate group, whereas Ni(2+) and Cu(2+) will preferably bind to N(3). Macrochelate formation for these metal ions is also predicted. The micro acidity constant for N(3)H(+) deprotonation in the (H???dImMP?H)(±) species (pk(a) 6.46) and the M(n+) -binding properties are of relevance for understanding the behavior of dImMP units present in DNA hairpins and metalated duplexes.     

Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming

Exposure of plants to chilling (low temperatures above freezing) limits growth and development in all environments outside the lowest latitudes. Cell ultrastructure and morphometric studies may allow associations to be made between chilling-induced changes at the ultrastructural level, molecular events and their physiological consequences. We examined changes in the shape, size and membrane organization of the organelles of mesophyll cells in Arabidopsis thaliana (Col 0), a cold-resistant species, after subjecting 6-week-old plants grown at normal growth temperatures to chilling (2.5–4°C; 14-h dark/10-h light cycle) for 6, 24 and 72 h and after a re-warming period of 50 h. No ultrastructural differences were seen in the first 6 h of chilling but after 24 h we observed swollen and rounded chloroplasts with larger starch grains and dilated thylakoids compared to control plants. By 72 h, chilling had resulted in a large accumulation of starch in chloroplasts, an apparent crowding of the cytosol and a lower abundance of peripheral reticulum than in the controls. The average area per chloroplast in cell sections increased after 72-h chilling while the number of chloroplasts remained the same. Ring-shaped and other morphologically aberrant mitochondria were present in significantly higher abundance in plants given 72 h chilling than in the controls. Plant re-warming for 50 h reduced chloroplast size to those of the controls and returned mitochondria to standard morphology, but peripheral reticulum remained less abundant than in plants never given a cold treatment. The near full return to normal ultrastructure upon plant re-warming indicates that the morphological changes may be part of acclimation to cold.