Unraveling the Enigma of Organismal Death: Insights, Implications, and Frontiers.

Significant knowledge gaps exist regarding the responses of cells, tissues, and organs to organismal death. Examining the survival mechanisms influenced by metabolism and environment, this research has the potential to transform regenerative medicine, redefine legal death, and provide insights into life's physiological limits, paralleling inquiries in embryogenesis.

Glucocorticoid receptor expression in multiple myeloma patients is a predictor of survival.

Multiple myeloma (MM) is a blood neoplasia characterized by abnormal proliferation of plasma cells. Various treatments such as stem cell transplant (SCT), proteasome inhibitors, immune-modulating drugs, monoclonal antibodies and selective inhibitors of nuclear export have been routinely used to treat MM. However, relapse and treatment resistance are common problems in MM patients. Treatments are enhanced by Dexamethasone (Dex), a synthetic steroid that activates the glucocorticoid receptor (GR) which leads to apoptosis. To evaluate the potential impact of GR expression on overall survival, MM patient data from the CoMMpass study of 650 patients were analyzed. Multivariate modeling results show that increased GR expression at diagnosis is associated with a decreased risk of dying relative to those with lower levels of expression.

Effects of selected deubiquitinating enzyme inhibitors on the proliferation and motility of lung cancer and mesothelioma cell lines.

The post­translational modification of proteins by ubiquitinating enzymes plays a central role in a number of cellular functions, such as cell proteolysis, DNA repair, and cell signaling and communication. Deubiquitinating enzymes (DUBs) disassemble ubiquitin chains and remove ubiquitin moieties from proteins. Targeting DUBs in cancer models has revealed an important role for these enzymes in tumorigenesis, and they therefore have emerged as attractive therapeutic targets. In the present study, the effects of three DUB inhibitors, PR­619, RA­9 and LDN­91946, on a non­small cell lung cancer cell line (A549) and a mesothelioma cell line (H2373) were investigated. PR­619 significantly inhibited cell adhesion and the proliferation of both cell lines. RA­9 exerted an inhibitory effect on the adhesion and proliferation of H2373 cells, whereas it had no effect on A549 cells. Notably, however, while PR­619 attenuated the proliferation of both cell lines, it exerted an opposite effect on cell motility; in the case of A549 cells, there was a significant increase in cell motility, while for the H2373 cells, there was a significant decrease. Furthermore, protein phosphorylation kinetic analyses revealed that the effects were cell line­specific. In H2373 cells, the phosphorylation of only one peptide corresponding to the P85A protein was significantly affected, and while LDN­91946 treatment increased phosphorylation, treatment with RA­9 or PR­619 decreased its phosphorylation compared to the DMSO control. By contrast, in the case of A549 cells, the phosphorylation of 21 peptides was significantly affected by the same compounds. In light of the potential for the negative side­effects of DUB inhibition, such as increased cancer cell motility, the data presented herein underscore the dire need for the development of specific DUB inhibitors and to elucidate the individual role of DUB family members in cancer biology before they can be specifically pharmacologically targeted.

Titanium as a modifier of the peri-implant microbiome structure.

BACKGROUND: Recent data support the implication of accelerated titanium dissolution products in peri-implantitis. It is unknown whether these dissolution products have an effect on the peri-implant microbiome, the target of existing peri-implantitis therapies. PURPOSE: This study assessed the relationship between the peri-implant microbiome, dissolved titanium levels, and peri-implantitis. MATERIALS AND METHODS: Clinical, microbiome, and titanium data were collected from a periodontal population having implants in function for 10 years. Clinical examinations were performed, and submucosal plaque samples were collected from the deepest site per implant. An aliquot of the sample was used for 16S rRNA gene sequencing, with the remainder analyzed for titanium quantity using mass spectrometry. Sequences were clustered into taxonomic units at 97% minimum sequence similarity using the QIIME pipeline approach. RESULTS: Fifteen implants were assessed. According to established case definitions, six had a diagnosis of peri-implantitis; nine were healthy. The genera Streptococcus, Prevotella and Haemophilus characterized peri-implant health. Peri-implantitis was associated with a marked increase in Veillonella. Quantities of dissolved titanium were identified in 40% of sites. Titanium presence was associated with peri-implant disease status (P = .02) and correlated to the first principal component of the microbiome (rho = 0.552) and its alpha-diversity (rho = -0.496). Canonical correlation analyses found that titanium levels, but not health or disease status of the implant, were significantly associated with the microbiota composition (P = .045). CONCLUSIONS: These findings suggest an association between titanium dissolution products and peri-implantitis and support a role for these products in modifying the peri-implant microbiome structure and diversity.

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.

Transcriptome changes after genome-wide admixture in invasive sculpins (Cottus).

Models on hybrid speciation assume that hybridization generates increased phenotypic variance that is utilized to invade new adaptive peaks. We test to what extent this prediction can be traced using gene expression data in the fish species Cottus perifretum and Cottus rhenanus as well as a natural hybrid lineage referred to as invasive sculpins. In addition, interspecies crosses were used to explore evolutionary trajectories from initial stages to the hybrid lineage. EST (expressed sequence tag) libraries were sequenced to design an oligonucleotide microarray that was calibrated for probe-specific differences in binding behaviour. Levels of gene expression divergence between species correlate with genetic divergence at neutral markers and, accordingly, invasive sculpins were intermediate between the parental species overall. However, the hybrid lineage is distinguished through unique patterns of gene expression that are enriched for biological functions which represent candidates for the fitness properties of invasive sculpins. We compare F(2) crosses with natural invasive sculpins to show that the variance in gene expression decreases in invasives. Moreover, few of the transgressive patterns of gene expression that distinguish invasives can be directly observed in F(2) crosses. This suggests that the invasive transcriptome was subject to secondary changes after admixture. The result is in line with an evolutionary process that reduces maladaptive variance and optimizes the phenotype of an emerging hybrid lineage.

Constructing a fish metabolic network model.

We report the construction of a genome-wide fish metabolic network model, MetaFishNet, and its application to analyzing high throughput gene expression data. This model is a stepping stone to broader applications of fish systems biology, for example by guiding study design through comparison with human metabolism and the integration of multiple data types. MetaFishNet resources, including a pathway enrichment analysis tool, are accessible at http://metafishnet.appspot.com.

Linking probe thermodynamics to microarray quantification.

Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models.

Generation, analysis and functional annotation of expressed sequence tags from the sheepshead minnow (Cyprinodon variegatus).

BACKGROUND: Sheepshead minnow (Cyprinodon variegatus) are small fish capable of withstanding exposure to very low levels of dissolved oxygen, as well as extreme temperatures and salinities. It is an important model in understanding the impacts and biological response to hypoxia and co-occurring compounding stressors such as polycyclic aromatic hydrocarbons, endocrine disrupting chemicals, metals and herbicides. Here, we initiated a project to sequence and analyze over 10,000 ESTs generated from the Sheepshead minnow (Cyprinodon variegatus) as a resource for investigating stressor responses. RESULTS: We sequenced 10,858 EST clones using a normalized cDNA library made from larval, embryonic and adult suppression subtractive hybridization-PCR (SSH) libraries. Post- sequencing processing led to 8,099 high quality sequences. Clustering analysis of these ESTs indentified 4,223 unique sequences containing 1,053 contigs and 3,170 singletons. BLASTX searches produced 1,394 significant (E-value < 10-5) hits and further Gene Ontology (GO) analysis annotated 388 of these genes. All the EST sequences were deposited by Expressed Sequence Tags database (dbEST) in GenBank (GenBank: GE329585 to GE337683). Gene discovery and annotations are presented and discussed. This set of ESTs represents a significant proportion of the Sheepshead minnow (Cyprinodon variegatus) transcriptome, and provides a material basis for the development of microarrays useful for further gene expression studies in association with stressors such as hypoxia, cadmium, chromium and pyrene.

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.

Functioning in situ: gene expression in Methylotenera mobilis in its native environment as assessed through transcriptomics.

Methylotrophs, organisms able to gain energy and carbon from compounds containing no carbon-carbon bonds, such as methane, methanol and methylated amines, are widespread in nature. However, knowledge of their nutrient preference and their metabolism is mostly based on experiments with cultures grown in defined laboratory conditions. Here, we use transcriptomics to explore the activity of one methylotroph, Methyotenera mobilis in its natural environment, lake sediment from which it has been previously isolated. Cells encapsulated in incubation cassettes were exposed to sediment conditions, with or without supplementation with a carbon/energy source (methylamine), and gene-expression patterns were compared for those cells to patterns for cells incubated in a defined medium supplemented with methylamine. A few specific trends in gene expression were observed at in situ conditions that may be of environmental significance, as follows. Expression of genes for the linear formaldehyde oxidation pathway linked to tetrahydromethanopterin increased, suggesting an important role for this pathway in situ, in contrast to laboratory condition culture, in which the cyclic ribulose monophosphate pathway seemed to be the major route for formaldehyde oxidation. Along with the ribulose monophosphate cycle that is also a major pathway for assimilating C(1) units, the methylcitric acid cycle seemd to be important in situ, suggesting that multicarbon compounds may be the natural carbon and/or energy substrates for M. mobilis, challenging the notion of an obligately methylotrophic lifestyle for this bacterium. We also detected a major switch in expression of genes responsible for the mode of motility between different conditions: from flagellum-enabled motility in defined medium to in situ expression of pili known to be involved in twitching motility and adherence. Overall, this study offers a novel approach for gaining insights into the lifestyle of individual microbes in their native environments.

An algorithm for the determination and quantification of components of nucleic acid mixtures based on single sequencing reactions.

BACKGROUND: Determination and quantification of nucleic acid components in a mixture is usually accomplished by microarray approaches, where the mixtures are hybridized against specific probes. As an alternative, we propose here that a single sequencing reaction from a mixture of nucleic acids holds enough information to potentially distinguish the different components, provided it is known which components can occur in the mixture. RESULTS: We describe an algorithm that is based on a set of linear equations which can be solved when the sequencing profiles of the individual components are known and when the number of sequenced nucleotides is larger than the number of components in the mixture. We have implemented the procedure for one type of sequencing approach, pyrosequencing, which produces a stepwise output of peaks that is particularly suitable for the procedure. As an example we use signature sequences from ribosomal RNA to distinguish and quantify several different species in a mixture. Using simulations, we show that the procedure may also be applicable for dideoxy sequencing on capillary sequencers, requiring only some instrument specific adaptations of protocols and software. CONCLUSION: The parallel sequencing approach described here may become a simple and cheap alternative to microarray experiments which aim at routine re-determination and quantification of known nucleic acid components from environmental samples or tissue samples.

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.