CTCF binding site classes exhibit distinct evolutionary, genomic, epigenomic and transcriptomic features

Background  

CTCF (CCCTC-binding factor) is an evolutionarily conserved zinc finger protein involved in diverse functions ranging from negative regulation of MYC, to chromatin insulation of the beta-globin gene cluster, to imprinting of the Igf2 locus. The 11 zinc fingers of CTCF are known to differentially contribute to the CTCF-DNA interaction at different binding sites. It is possible that the differences in CTCF-DNA conformation at different binding sites underlie CTCF's functional diversity. If so, the CTCF binding sites may belong to distinct classes, each compatible with a specific functional role.     

Biochemical characterization of a strictly specific beta-galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum larvae

A beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum L. larvae was purified by chromatography on ion exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. Beta‐galactosidase was a monomeric protein with a molecular weight of 62 kDa based on its mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 60 kDa based on gel filtration. Maximal enzyme activity occurred at 55°C and pH 5.0. The purified beta‐galactosidase was stable at 37°C and its pH stability was in the range of 4.6–6.0. Beta‐galactosidase was highly specific for the beta‐d ‐galactosyl residue and beta‐(1‐4) linkage. The catalytic efficiency (V_max/K_m) values for p‐nitrophenyl‐beta‐d ‐galactopyranoside, beta‐d ‐galactosyl(1‐4)‐d ‐glucose (lactose), beta‐d ‐galactosyl(1‐4)‐d ‐galactose and beta‐d ‐galactosyl(1‐4)‐beta‐d ‐galactosyl(1‐4)‐d ‐glucose were, respectively, 72.95, 10.97, 20.74 and 12.73. 5,5‐Dithio‐bis(2‐nitrobenzoate) and sodium dodecyl sulfate inhibited completely the beta‐galactosidase activity. The enzyme was capable of catalyzing transgalactosylation reactions. The yield of galactosylation of 2‐phenylethanol (43%), catalyzed by the beta‐galactosidase in the presence of lactose as galactosyl donor, is higher than those reported previously with conventional sources of beta‐galactosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transgalactosylation reactions (pH 6.0).     

Ecoinvent 3: assessing water use in LCA and facilitating water footprinting

Purpose

Water footprinting and the assessment of water use in life cycle assessment have become of major interest in sustainability assessments. Various initiatives for combining water resource issues with consumption of products and services have been initiated in the last decade. However, comprehensive databases fulfilling the requirements for addressing these issues have been lacking and are necessary to facilitate efficient and consistent assessments of products and services. To this purpose, ecoinvent focused on integrating appropriate water use data into version 3, since previously water use data has been inconsistently reported and some essential flows were missing. This paper describes the structure of the water use data in ecoinvent, how the data has been compiled and the way it can be used for water footprinting.

Methods

The main changes required for proper assessment of water use are the addition of environmental and product flows in order to allow a water balance over each process. This is in accordance with the strict paradigm in ecoinvent 3 to focus on mass balances, which requires the inclusion of water contents of all products (also for e.g. waste water flows), as well as emissions of water to soil, air and various water bodies. Water inputs from air (e.g. rainwater harvesting) is introduced but is not yet used by any activity.

Results and discussion

Ecoinvent version 3.1 consistently includes the relevant flows to address water use in life cycle assessment (LCA) and calculate water footprints on the product level for most processes including uncertainty information. Although some problems regarding data quality and spatial resolution remain, this is an important step forward and can limit efforts for detailed data collection to the most sensitive processes in the product system. With the combination of data on water use and emissions to water for each process, concentration and corresponding water classes can also be calculated and assessed with existing impact assessment methods.

Conclusions

This comprehensive collection of water use data on the process level facilitates the proper assessment of water use within an LCA and water footprints beyond agricultural production. Especially in LCA, but also in tools for eco-design and specific water footprint, this data is essential and leads to a cost-efficient way of assessing consumption choices and product design decisions with full transparency. It enhances the effectiveness of investing in data collection by performing sensitivity analyses using ecoinvent data to identify the most relevant flows and processes.

     

Hierarchical models for combining ecological and case-control data

The ecological study design suffers from a broad range of biases that result from the loss of information regarding the joint distribution of individual-level outcomes, exposures, and confounders. The consequent nonidentifiability of individual-level models cannot be overcome without additional information; we combine ecological data with a sample of individual-level case-control data. The focus of this article is hierarchical models to account for between-group heterogeneity. Estimation and inference pose serious computational challenges. We present a Bayesian implementation based on a data augmentation scheme where the unobserved data are treated as auxiliary variables. The methods are illustrated with a dataset of county-specific infant mortality data from the state of North Carolina.     

Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem

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The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.     

Isolation of lactoferrin from milk of different species: calorimetric and antimicrobial studies

Lactoferrin (LF) is an iron-binding glycoprotein found in different biological fluids of mammals and in neutrophils. It has been proposed to be involved in many functions, including protection from pathogens. In this work, purification of lactoferrin using an ion-exchange chromatography (SP-Sepharose) was attempted for the milk of the following animals: sheep (Ovis aries), goat (Capra hircus), camel (Camelus bactrianus), alpaca (Lama pacos), elephant (Elephas maximus) and grey seal (Halichoerus grypus), as well as human (Homo sapiens). Lactoferrin was identified in all the milks apart from that from grey seal. The thermal stability of the purified lactoferrins, in their native and iron-saturated forms, was studied by differential scanning calorimetry (DSC). Maximum temperature, onset temperature and enthalpy change of denaturation were higher when lactoferrins were saturated with iron than in their native form, indicating an increase in the stability of the protein structure upon iron-binding. Human lactoferrin was found to be the most heat-resistant and the other lactoferrins presented different degrees of thermoresistance, that of elephant being the least resistant. The antimicrobial activity of the different isolated lactoferrins was investigated against Escherichia coli 0157:H7. The minimal inhibitory concentrations (MICs) were determined by measuring the absorbance at 620 nm. The minimum bactericidal concentrations (MBCs) were also measured and it was found that camel lactoferrin was the most active lactoferrin against E. coli 0157:H7, whereas alpaca and human lactoferrins were the least active.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.