Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system

The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Incidental and Intentional Learning of Verbal Episodic Material Differentially Modifies Functional Brain Networks

Learning- and memory-related processes are thought to result from dynamic interactions in large-scale brain networks that include lateral and mesial structures of the temporal lobes. We investigate the impact of incidental and intentional learning of verbal episodic material on functional brain networks that we derive from scalp-EEG recorded continuously from 33 subjects during a neuropsychological test schedule. Analyzing the networks'' global statistical properties we observe that intentional but not incidental learning leads to a significantly increased clustering coefficient, and the average shortest path length remains unaffected. Moreover, network modifications correlate with subsequent recall performance: the more pronounced the modifications of the clustering coefficient, the higher the recall performance. Our findings provide novel insights into the relationship between topological aspects of functional brain networks and higher cognitive functions.     

Impact of Lauric Arginate Application Form on its Antimicrobial Activity in Meat Emulsions

Lauric arginate (LAE) is a food-grade cationic surfactant that is highly active against a wide range of food pathogens (Listeria monocytogenes, Salmonella, and Escherichia coli) and food spoilers (Lactobacilli, yeast, and molds). The antimicrobial efficacy of LAE in compositionally complex environments is likely to be negatively impacted by its interactions with food ingredients. Therefore, we investigated different application systems of LAE and their impact on its antimicrobial efficacy when added to “Lyoner style” sausages. LAE was applied as a powder, aqueous solution, in oil-in-water emulsions with different droplet sizes, and as solid lipid particles (SLP) with different droplet sizes. Structures of the systems were identified by optical microscopy, differential scanning calorimetry (DSC) and static light scattering. A recontamination on the surface of sliced sausages was simulated using Listeria innocua as the target organism (2 log colony forming units (CFU)/slice), and the antimicrobial impact of 1,000, 1,500 and 2,000 μg/g applied LAE in the sausage was examined by growth curves. A modeling of the CFU-time relationship was carried out to provide a better evaluation of the antimicrobial activity of LAE. Finally, we carried out an isothermal titration calorimetry (ITC) analysis to simulate the interactions between LAE and proteins in the sausage matrix. Results revealed that the application systems differed in their surface area and, therefore, showed different antimicrobial activities when incorporated into sausage. The study demonstrated that the SLP and emulsions as LAE application systems increased the antimicrobial activity against microbial growth on the surface of sliced “Lyoner style” sausages.     

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (< 50 °C), the enzymatic activity of the protein strongly decreased during storage at 60 °C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20 °C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.     

Detection of dairy fouling: Combining ultrasonic measurements and classification methods

Fouling and cleaning in heat exchangers are severe and costly (up to 0.3% of gross national product) issues in dairy and food processing. Therefore, reducing cleaning time and cost is urgently needed. In this study, two classification methods [artificial neural network (ANN) and support vector machine (SVM)] for detecting protein and mineral fouling presence and absence based on ultrasonic measurements were presented and compared. ANN is based on a multilayer perceptron feed forward neural network, whereas SVM is based on clustering between fouling and no fouling using a hyperplane. When both fouling types (1239 datasets) were combined, ANN showed an accuracy of 71.9% while SVM displayed an accuracy of 97.6%. Separate fouling detection of mineral/protein fouling by ANN/SVM was comparable: dependent on fouling type detection accuracies of 100% (protein fouling, ANN and SVM), and 98.2% (SVM), and 93.5% (ANN) for mineral fouling was reached. It was shown that it was possible to detect fouling presence and absence offline in a static setup using ultrasonic measurements in combination with a classification method. This study proved the applicability of combining classification methods and fouling measurements to take a step toward reducing cleaning costs and time.     

RAIphy: Phylogenetic classification of metagenomics samples using iterative refinement of relative abundance index profiles

Background  

Computational analysis of metagenomes requires the taxonomical assignment of the genome contigs assembled from DNA reads of environmental samples. Because of the diverse nature of microbiomes, the length of the assemblies obtained can vary between a few hundred bp to a few hundred Kbp. Current taxonomic classification algorithms provide accurate classification for long contigs or for short fragments from organisms that have close relatives with annotated genomes. These are significant limitations for metagenome analysis because of the complexity of microbiomes and the paucity of existing annotated genomes.     

Insights into chemotaxonomic composition and carbon cycling of phototrophic communities in an artesian sulfur-rich spring (Zodletone, Oklahoma, USA), a possible analog for ancient microbial mat systems

Zodletone spring in Oklahoma is a unique environment with high concentrations of dissolved-sulfide (10 mm) and short-chain gaseous alkanes, exhibiting characteristics that are reminiscent of conditions that are thought to have existed in Earth's history, in particular the late Archean and early-to-mid Proterozoic. Here, we present a process-oriented investigation of the microbial community in two distinct mat formations at the spring source, (1) the top of the sediment in the source pool and (2) the purple streamers attached to the side walls. We applied a combination of pigment and lipid biomarker analyses, while functional activities were investigated in terms of oxygen production (microsensor analysis) and carbon utilization ((13)C incorporation experiments). Pigment analysis showed cyanobacterial pigments, in addition to pigments from purple sulfur bacteria (PSB), green sulfur bacteria (GSB) and Chloroflexus-like bacteria (CLB). Analysis of intact polar lipids (IPLs) in the source sediment confirmed the presence of phototrophic organisms via diacylglycerol phospholipids and betaine lipids, whereas glyceroldialkylglyceroltetraether additionally indicated the presence of archaea. No archaeal IPLs were found in the purple streamers, which were strongly dominated by betaine lipids. (13)C-bicarbonate- and -acetate-labeling experiments indicated cyanobacteria as predominant phototrophs in the source sediment, carbon was actively fixed by PSB/CLB/GSB in purple streamers by using near infrared light. Despite the presence of cyanobacteria, no oxygen could be detected in the presence of light, suggesting anoxygenic photosynthesis as the major metabolic process at this site. Our investigations furthermore indicated photoheterotrophy as an important process in both habitats. We obtained insights into a syntrophically operating phototrophic community in an ecosystem that bears resemblance to early Earth conditions, where cyanobacteria constitute an important contributor to carbon fixation despite the presence of high sulfide concentrations.     

UNiquant, a program for quantitative proteomics analysis using stable isotope labeling

Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for postmeasurement normalization of peptide ratios, which is required by the other programs.