Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli.

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA ß-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

WIF1 Suppresses the Generation of Suprabasal Cells in Acanthotic Skin and Growth of Basal Cell Carcinomas upon Forced Overexpression.

We analyzed the role of WIF1 in normal and acanthotic epidermis of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans-retinoic acid (ATRA)-treated and basal cell carcinoma (BCC)-bearing mice. WIF1 protein is located in the follicular infundibulum and interfollicular epidermis (IFE) in murine back skin. Within the hyperplastic epidermis of TPA- or ATRA-treated or BCC-bearing murine skin, WIF1 and Keratin 10 overlap in Ki67⁻ suprabasal layers, while basal epidermal layers expressing Ki67, and BCCs expressing Wif1 mRNA, are free of WIF1 protein. This is similar in human skin, with the exception that WIF1 protein is found in single Ki67⁻ basal epidermal cells in normal skin and additionally in Ki67+ cells in acanthotic skin. Wif1-deficiency enhances acanthosis of the murine BCC-associated epidermis, which is accompanied by an increase of Ki67+ and of Sca-1+ basal cells. WIF1 overexpression in allografted BCC-derived keratinocytes prevents growth and keratinization, involving enhanced phosphorylation of protein kinase C and extracellular signal-regulated kinase 1 and arguably factors secreted by the in vivo environment. In summary, WIF1 protein marks suprabasal layers in the normal IFE. It is also present in the epidermis overlaying BCCs where it diminishes proliferation of basal cells and production of differentiating suprabasal cells. In addition, WIF1 can prevent proliferation and keratinization of BCC-related keratinocytes.

Patched knockout mouse models of Basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common human tumor. Mutations in the hedgehog (HH) receptor Patched (PTCH) are the main cause of BCC. Due to their high and increasing incidence, BCC are becoming all the more important for the health care system. Adequate animal models are required for the improvement of current treatment strategies. A good model should reflect the situation in humans (i.e., BCC initiation due to Ptch mutations on an immunocompetent background) and should allow for (i) BCC induction at a defined time point, (ii) analysis of defined BCC stages, and (iii) induction of BCC in 100% of animals. In addition, it should be easy to handle. Here, we compare several currently existing conventional and conditional Ptch knockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditional Ptch(flox/flox) mice and the K5-Cre-ER(T+/-) driver.

A microfluidic array with cellular valving for single cell co-culture.

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (∼99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (∼70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.

Circulating levels of Clara cell protein 16 but not surfactant protein D identify and quantify lung damage in patients with multiple injuries.

BACKGROUND: Almost 60% of all patients with severe multiple injuries sustain severe chest trauma with aggravating effect on morbidity and mortality. Diagnosis of lung contusion is performed by early posttraumatic multislice computed tomography. Because this diagnostic procedure requires time, resources, and exposure to radiation, a noninvasive approach with easy follow-up measurements is warranted. METHODS: Serum levels of Clara cell protein 16 (CC16) and surfactant protein D as lung-specific biomarkers were obtained on admission from 104 patients with multiple injuries using enzyme-linked immunosorbent assay technique. Patients were divided into those with severe lung injury ([LI]; n = 68) and without LI (NLI; n = 36). Nonsmoking healthy volunteers served as controls. In addition, volume of lung contusions were calculated planimetrically on serial multislice computed tomography scans obtained after admission. Factors influencing CC16 serum levels were determined in uni- and multivariate analyses, and Spearman rank coefficients were calculated for correlations. RESULTS: Patients with LI showed a significant (p < 0.05) elevation of median CC16 levels (10.2 ng/mL) compared with NLI patients (5.4 ng/mL) and controls (5.2 ng/mL). Serum CC16 levels correlated with the volume of lung contusions (r = 0.78, p < 0.0001) and were not influenced by overall injury severity, age, gender, or preclinical ventilation. In contrast, circulating surfactant protein D levels were not associated with the presence of LI or the extent of lung contusions. CONCLUSIONS: Our results advocate CC16 as a potential biomarker for LI in severely injured patients because of its high correlation with the volume of contused lung parenchyma. Therefore, this parameter may allow a specified initial treatment of patients with multiple injuries.

Temperature gradient focusing in miniaturized free-flow electrophoresis devices.

Temperature gradient focusing is a method to separate and focus any charged analytes even without accessible isoelectric point, and has been already widely used in CE. In this paper, we demonstrate the application of temperature gradient focusing to free-flow electrophoresis. Besides focusing and separation experiments of proteins, the stability of the temperature gradient under flow conditions and the temperature dependence of fluorescence dyes have also been investigated.

Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels.

A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip.

Isotachophoretic free-flow electrophoretic focusing and SERS detection of myoglobin inside a miniaturized device.

A new approach for label-free optical sample detection via surface enhanced Raman spectroscopy (SERS) inside a free-flow electrophoresis (FFE) chip is presented in this work.

Persistent radicals of trivalent tin and lead.

In this report we present synthetic, crystallographic, and new electron paramagnetic resonance (EPR) spectroscopic work that shows that the synthetic route leading to the recently reported, first persistent plumbyl radical *PbEbt3 (Ebt = ethylbis(trimethylsilyl)silyl), that is, the oxidation of the related PbEbt3-anion, was easily extended to the synthesis of other persistent molecular mononuclear radicals of lead and tin. At first, various novel solvates of homoleptic potassium metallates KSnHyp3 (4a), KPbHyp3 (3a), KSnEbt3 (4b), KPbIbt3 (3c), and KSnIbt3 (4c) (Hyp = tris(trimethylsilyl)silyl, Ibt = isopropylbis(trimethylsilyl)silyl), as well as some heteroleptic metallates, such as [Li(OEt2)2][Sn(n)BuHyp2] (3d), [Li(OEt2)2][Pb(n)BuHyp2] (4d), [Li(thf)4][PbPhHyp2] (3e), and [K(thf)7][PbHyp2{N(SiMe3)2}] (3f), were synthesized and crystallographically characterized. Through oxidation by tin(II) and lead(II) bis(trimethylsilyl)amides or the related 2,6-di-tert-butylphenoxides, they had been oxidized to yield in most cases the corresponding radicals. Five novel persistent homoleptically substituted radicals, that is, *SnHyp3 (2a), *PbHyp3 (1a), *SnEbt3 (2b), *SnIbt3 (2c), and *PbIbt3 (1c), had been characterized by EPR spectroscopy. The stannyl radicals 2a and 2c as well as the plumbyl radical 1c were isolated as intensely colored crystalline compounds and had been characterized by X-ray diffraction. Persistent heteroleptically substituted radicals such as *PbHyp2Ph (1e) or *PbHyp2Et (1g) had also been generated, and some selected EPR data are given for comparison. The plumbyl radicals *PbR3 exhibit a clean monomolecular decay leading to the release of a temperature-dependent stationary concentration of branched silyl radicals. They may thus serve as tunable sources of these reactive species that may be utilized as reagents for mild radical silylations and/or as initiators for radical polymerizations. We present EPR-spectroscopic investigations for the new tin- and lead-containing compounds giving detailed insights into their electronic and geometric structure in solution, as well as structural studies on the crystalline state of the radicals, some of their anionic precursors, and some side-products.

Exotropias / Exotropia

O Autor discute a exotropia intermitente demonstando os passos necesários para o seu diagnóstico e tratamento. O Controle da exotropia intermitente é feito com recursos clínicos que visam combater a supressäo e aumentar a convergência acomodativa para obter um bom desenvolvimento da visiao binocular e após 6 anos a cirurgia que e feita em duas etapas. Na primeira obtem-se uma hipercorreçäo de 20 diptrias para criar diplopia e na segunda realiza-se uma pequena miotomia para relaxar o RM