In support of descriptive studies; relevance to translational research

The contemporary scientific establishment equates hypothesis testing to good science. This stance bypasses the preliminary need to identify a worthwhile hypothesis through rigorous observation of natural processes. If alleviation of human suffering is claimed as the goal of a scientific undertaking, it would be unfair to test a hypothesis whose relevance to human disease has not been satisfactorily proven. Here, we argue that descriptive investigations based on direct human observation should be highly valued and regarded essential for the selection of worthwhile hypotheses while the pursuit of costly scientific investigations without such evidence is a desecration of the cause upon which biomedical research is grounded

Ensemble Modeling for Aromatic Production in Escherichia coli

Ensemble Modeling (EM) is a recently developed method for metabolic modeling, particularly for utilizing the effect of enzyme tuning data on the production of a specific compound to refine the model. This approach is used here to investigate the production of aromatic products in Escherichia coli. Instead of using dynamic metabolite data to fit a model, the EM approach uses phenotypic data (effects of enzyme overexpression or knockouts on the steady state production rate) to screen possible models. These data are routinely generated during strain design. An ensemble of models is constructed that all reach the same steady state and are based on the same mechanistic framework at the elementary reaction level. The behavior of the models spans the kinetics allowable by thermodynamics. Then by using existing data from the literature for the overexpression of genes coding for transketolase (Tkt), transaldolase (Tal), and phosphoenolpyruvate synthase (Pps) to screen the ensemble, we arrive at a set of models that properly describes the known enzyme overexpression phenotypes. This subset of models becomes more predictive as additional data are used to refine the models. The final ensemble of models demonstrates the characteristic of the cell that Tkt is the first rate controlling step, and correctly predicts that only after Tkt is overexpressed does an increase in Pps increase the production rate of aromatics. This work demonstrates that EM is able to capture the result of enzyme overexpression on aromatic producing bacteria by successfully utilizing routinely generated enzyme tuning data to guide model learning

Repulsive Forces Between Looping Chromosomes Induce Entropy-Driven Segregation

One striking feature of chromatin organization is that chromosomes are compartmentalized into distinct territories during interphase, the degree of intermingling being much smaller than expected for linear chains. A growing body of evidence indicates that the formation of loops plays a dominant role in transcriptional regulation as well as the entropic organization of interphase chromosomes. Using a recently proposed model, we quantitatively determine the entropic forces between chromosomes. This Dynamic Loop Model assumes that loops form solely on the basis of diffusional motion without invoking other long-range interactions. We find that introducing loops into the structure of chromatin results in a multi-fold higher repulsion between chromosomes compared to linear chains. Strong effects are observed for the tendency of a non-random alignment; the overlap volume between chromosomes decays fast with increasing loop number. Our results suggest that the formation of chromatin loops imposes both compartmentalization as well as order on the system without requiring additional energy-consuming processes

Artificial Modulation of the Gating Behavior of a K+ Channel in a KvAP-DNA Chimera

We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel

Lethal Thermal Impact at Periphery of Pyroclastic Surges: Evidences at Pompeii

Background: The evaluation of mortality of pyroclastic surges and flows (PDCs) produced by explosive eruptions is a major goal in risk assessment and mitigation, particularly in distal reaches of flows that are often heavily urbanized. Pompeii and the nearby archaeological sites preserve the most complete set of evidence of the 79 AD catastrophic eruption recording its effects on structures and people. Methodology/Principal Findings: Here we investigate the causes of mortality in PDCs at Pompeii and surroundings on the bases of a multidisciplinary volcanological and bio-anthropological study. Field and laboratory study of the eruption products and victims merged with numerical simulations and experiments indicate that heat was the main cause of death of people, heretofore supposed to have died by ash suffocation. Our results show that exposure to at least 250uC hot surges at a distance of 10 kilometres from the vent was sufficient to cause instant death, even if people were sheltered within buildings. Despite the fact that impact force and exposure time to dusty gas declined toward PDCs periphery up to the survival conditions, lethal temperatures were maintained up to the PDCs extreme depositional limits. Conclusions/Significance: This evidence indicates that the risk in flow marginal zones could be underestimated by simply assuming that very thin distal deposits, resulting from PDCs with poor total particle load, correspond to negligible effects. Therefore our findings are essential for hazard plans development and for actions aimed to risk mitigation at Vesuvius an

I-Motif Structures Formed in the Human c-MYC Promoter Are Highly Dynamic–Insights into Sequence Redundancy and I-Motif Stability

The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3′ consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+–cytosine base pairs are not always necessary for an intramolecular I-motif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif

ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-κB-dependent manner

<p>Abstract</p> <p>Background</p> <p>Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.</p> <p>Methods</p> <p>We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.</p> <p>Results</p> <p>Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.</p> <p>Conclusions</p> <p>We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.</p

T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein

Hi-C-constrained physical models of human chromosomes recover functionally-related properties of genome organization

Combining genome-wide structural models with phenomenological data is at the forefront of efforts to understand the organizational principles regulating the human genome. Here, we use chromosome-chromosome contact data as knowledge-based constraints for large-scale three-dimensional models of the human diploid genome. The resulting models remain minimally entangled and acquire several functional features that are observed in vivo and that were never used as input for the model. We find, for instance, that gene-rich, active regions are drawn towards the nuclear center, while gene poor and lamina associated domains are pushed to the periphery. These and other properties persist upon adding local contact constraints, suggesting their compatibility with non-local constraints for the genome organization. The results show that suitable combinations of data analysis and physical modelling can expose the unexpectedly rich functionally-related properties implicit in chromosome-chromosome contact data. Specific directions are suggested for further developments based on combining experimental data analysis and genomic structural modelling

<em>Enterococcus faecalis</em> Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

Background: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Results: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-kappa B inflammatory response as well as impaired DNA damage response and cell cycle control gene expression