Improvement of Acetaldehyde Production in Zymomonas mobilis by Engineering of Its Aerobic Metabolism.

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers.

Cooperative and distinct functions of MK2 and MK3 in the regulation of the macrophage transcriptional response to lipopolysaccharide.

The p38MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2-/- or MK2/3-/- animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-ß. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-ß. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBß, reflecting the substantial plasticity of the response of BMDM to LPS.

IL-1ß and TNFα Differentially Influence NF-κB Activity and FasL-Induced Apoptosis in Primary Murine Hepatocytes During LPS-Induced Inflammation.

Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1ß, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1ß induces increased caspase-3 activity but keeps cells alive. We now report that IL-1ß and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1ß and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1ß and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection.

In silico evaluation of geometry variations with respect to the thermal spread during coagulation of egg white using bipolar vessel sealing instruments.

BACKGROUND: Bipolar vessel sealing is an efficient electrosurgical procedure for the occlusion of blood vessels particularly during minimally invasive surgery. Reliable knowledge of the thermal spread is crucial for a safe application of bipolar vessel sealing instruments when operating close to thermo-sensitive structures, such as nerves. The evolution of the thermal spread over time and space depends on a variety of parameters, such as the biological tissue, the energy applied to the tissue, and the geometry of the vessel sealing instrument. Mathematical modeling has proven useful for the prediction of the thermal spread. It is, thus, a promising tool for the systematic analysis of the influence of geometrical changes on the thermal spread. RESULTS: We present an experimentally validated in silico study to evaluate the impact of geometry variations on the progression of chicken egg white coagulation and the final shape of coagulated egg white as an approximation of the temporal and spatial evolution of the thermal spread during bipolar vessel sealing. Egg white has similar thermal and electrical properties to human tissue, with the advantage being that the spatial and temporal evolution of the thermal spread can be visually gauged. The simulations were performed using a mathematical model based on the finite element analysis of chicken egg white. The progression of egg white coagulation was predicted for two different peak voltages and various electrode geometries. Starting with two planar electrodes, one electrode was gradually changed to adopt a wedge shape. These changes to the geometry showed a distinct influence on the progression of egg white coagulation in the simulations. The predictions were successfully validated using an experimental setup with two different electrodes representing the extreme geometries. DISCUSSION: The predicted spatial temperature distributions were experimentally validated for two geometries. Our simulation study shows that the geometry has a pronounced influence on the thermal spread and, thus, is a suitable parameter to reduce thermal damage. The in silico optimization of instrument designs is a suitable tool to accelerate the development of new vessel sealing instruments, with only a few promising designs having to be tested as prototypes.

Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages.

Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.