Cryptic sequence features in the active postmortem transcriptome.

BACKGROUND: Our previous study found that more than 500 transcripts significantly increased in abundance in the zebrafish and mouse several hours to days postmortem relative to live controls. The current literature suggests that most mRNAs are post-transcriptionally regulated in stressful conditions. We rationalized that the postmortem transcripts must contain sequence features (3- to 9- mers) that are unique from those in the rest of the transcriptome and that these features putatively serve as binding sites for proteins and/or non-coding RNAs involved in post-transcriptional regulation. RESULTS: We identified 5117 and 2245 over-represented sequence features in the mouse and zebrafish, respectively, which represents less than 1.5% of all possible features. Some of these features were disproportionately distributed along the transcripts with high densities in the 3' untranslated regions of the zebrafish (0.3 mers/nt) and the open reading frames of the mouse (0.6 mers/nt). Yet, the highest density (2.3 mers/nt) occurred in the open reading frames of 11 mouse transcripts that lacked 3' or 5' untranslated regions. These results suggest the transcripts with high density of features might serve as 'molecular sponges' that sequester RNA binding proteins and/or microRNAs, and thus indirectly increase the stability and gene expression of other transcripts. In addition, some of the features were identified as binding sites for Rbfox and Hud proteins that are also involved in increasing transcript stability and gene expression. CONCLUSIONS: Our results are consistent with the hypothesis that transcripts involved in responding to extreme stress, such as organismal death, have sequence features that make them different from the rest of the transcriptome. Some of these features serve as putative binding sites for proteins and non-coding RNAs that determine transcript stability and fate. A small number of the transcripts have high density sequence features, which are presumably involved in sequestering RNA binding proteins and microRNAs and thus preventing regulatory interactions among other transcripts. Our results provide baseline data on post-transcriptional regulation in stressful conditions that has implications for regulation in disease, starvation, and cancer.

Developing a low-cost milliliter-scale chemostat array for precise control of cellular growth.

BACKGROUND: Multiplexed milliliter-scale chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for carrying out evolution experiments. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters - the accuracy, precision, and operational range of flow rate - are not explicitly characterized. METHODS: Here we re-purpose a published multiplexed culturing device to develop a multiplexed milliliter-scale chemostat. Flow rates for eight chambers can be independently controlled to a wide range, corresponding to population doubling times of 3~13 h, without the use of expensive feedback systems. RESULTS: Flow rates are precise, with the maximal coefficient of variation among eight chambers being less than 3%. Flow rates are accurate, with average flow rates being only slightly below targets, i.e., 3%-6% for 13-h and 0.6%-1.0% for 3-h doubling times. This deficit is largely due to evaporation and should be correctable. We experimentally demonstrate that our device allows accurate and precise quantification of population phenotypes. CONCLUSIONS: We achieve precise control of cellular growth in a low-cost milliliter-scale chemostat array, and show that the achieved precision reduces the error when measuring biological processes.

Gene Meter: Accurate abundance calculations of gene expression.

We previously reported that thousands of transcripts in the mouse and zebrafish significantly increased in abundance in a time series spanning from life to several days after death. Transcript abundances were determined by: calibrating each microarray probe using a dilution series of pooled RNAs, fitting the probe-responses to adsorption models, and back-calculating abundances using the probe signal intensity of a sample and the best fitting model. The accuracy of the abundance measurements was not assessed in our previous study because individual transcript concentrations in the calibration pool were not known. Accurate transcript abundances are highly desired for modeling the dynamics of biological systems and investigating how systems respond to perturbations. In this study, we show that accurate transcript abundances can be determined by calibrating the probes using a calibration pool of transcripts with known concentrations. Instructions for determining accurate transcript abundances using the Gene Meter approach are provided.

Gene expression in the twilight of death: The increase of thousands of transcripts has implications to transplantation, cancer, and forensic research.

After a vertebrate dies, many of its organ systems, tissues, and cells remain functional while its body no longer works as a whole. We define this state as the "twilight of death" - the transition from a living body to a decomposed corpse. We claim that the study of the twilight of death is important to ethical, legal and medical science. We examined gene expression at the twilight of death in the zebrafish and mouse reaching the conclusion that apparently thousands of transcripts significantly increase in abundance from life to several hours/days postmortem relative to live controls. Transcript dynamics of different genes provided "proof-of-principle" that models accurately predict an individual's elapsed-time-of-death (i.e. postmortem interval). While many transcripts were associated with survival and stress compensation, others were associated with epigenetic factors, developmental control, and cancer. Future studies are needed to determine whether the high incidence of cancer in transplant recipients is due to the postmortem processes in donor organs.

Distinctive thanatomicrobiome signatures found in the blood and internal organs of humans.

According to the Human Microbiome Project, 90% of the cells in a healthy adult body are microorganisms. What happens to these cells after human host death, defined here as the thanatomicrobiome (i.e., thanatos-, Greek defn., death), is not clear. To fill the void, we examined the thanatomicrobiome of the spleen, liver, brain, heart and blood of human cadavers. These organs are thought to be devoid of microorganisms in a healthy adult host. We report that the thanatomicrobiome was highly similar among organ tissues from the same cadaver but very different among the cadavers possibly due to differences in the elapsed time since death and/or environmental factors. Isolation of microbial DNA from cadavers is known to be a challenge. We compared the effectiveness of two methods by amplifying the 16S rRNA genes and sequencing the amplicons from four cadavers. Paired comparisons revealed that the conventional DNA extraction method (bead-beating in phenol/chloroform/bead-beating followed by ethanol precipitation) yielded more 16S rRNA amplicons (28 of 30 amplicons) than a second method (repeated cycles of heating/cooling followed by centrifugation to remove cellular debris) (19 of 30 amplicons). Shannon diversity index of the 16S rRNA genes revealed no significant difference by extraction method. The present report provides a proof of principle that the thanatomicrobiome may be an efficient biomarker to study postmortem transformations of cadavers.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Scanner calibration revisited.

BACKGROUND: Calibration of a microarray scanner is critical for accurate interpretation of microarray results. Shi et al. (BMC Bioinformatics, 2005, 6, Art. No. S11 Suppl. 2.) reported usage of a Full Moon BioSystems slide for calibration. Inspired by the Shi et al. work, we have calibrated microarray scanners in our previous research. We were puzzled however, that most of the signal intensities from a biological sample fell below the sensitivity threshold level determined by the calibration slide. This conundrum led us to re-investigate the quality of calibration provided by the Full Moon BioSystems slide as well as the accuracy of the analysis performed by Shi et al. METHODS: Signal intensities were recorded on three different microarray scanners at various photomultiplier gain levels using the same calibration slide from Full Moon BioSystems. Data analysis was conducted on raw signal intensities without normalization or transformation of any kind. Weighted least-squares method was used to fit the data. RESULTS: We found that initial analysis performed by Shi et al. did not take into account autofluorescence of the Full Moon BioSystems slide, which led to a grossly distorted microarray scanner response. Our analysis revealed that a power-law function, which is explicitly accounting for the slide autofluorescence, perfectly described a relationship between signal intensities and fluorophore quantities. CONCLUSIONS: Microarray scanners respond in a much less distorted fashion than was reported by Shi et al. Full Moon BioSystems calibration slides are inadequate for performing calibration. We recommend against using these slides.

Effect of high pressure and reversed micelles on the fluorescent proteins.

Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure.

An algorithm and program for finding sequence specific oligonucleotide probes for species identification.

BACKGROUND: The identification of species or species groups with specific oligo-nucleotides as molecular signatures is becoming increasingly popular for bacterial samples. However, it shows also great promise for other small organisms that are taxonomically difficult to tract. RESULTS: We have devised here an algorithm that aims to find the optimal probes for any given set of sequences. The program requires only a crude alignment of these sequences as input and is optimized for performance to deal also with very large datasets. The algorithm is designed such that the position of mismatches in the probes influences the selection and makes provision of single nucleotide outloops. Program implementations are available for Linux and Windows.