Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Longitudinal exchange: an alternative strategy towards quantification of dynamics parameters in ZZ exchange spectroscopy

Longitudinal exchange experiments facilitate the quantification of the rates of interconversion between the exchanging species, along with their longitudinal relaxation rates, by analyzing the time-dependence of direct correlation and exchange cross peaks. Here we present a simple and robust alternative to this strategy, which is based on the combination of two complementary experiments, one with and one without resolving exchange cross peaks. We show that by combining the two data sets systematic errors that are caused by differential line-broadening of the exchanging species are avoided and reliable quantification of kinetic and relaxation parameters in the presence of additional conformational exchange on the ms–μs time scale is possible. The strategy is applied to a bistable DNA oligomer that displays different line-broadening in the two exchanging species

Functional Diversity of Human Basic Helix-Loop-Helix Transcription Factor TCF4 Isoforms Generated by Alternative 5′ Exon Usage and Splicing

BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2) is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG). While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence about the functional diversity of the alternative TCF4 protein isoforms

Inactivation of Listeria monocytogenes in raw and hot smoked trout fillets by high hydrostatic pressure processing combined with liquid smoke and freezing

High hydrostatic pressure (HHP; 200 MPa for 15 min), liquid smoke (0.50%, v/v) and freezing (−80 °C, overnight) was used to eliminate Listeria monocytogenes in BHI broth, raw and smoked trout. The bactericidal effect of liquid smoke solutions (L9 and G6), HHP and their combinations was evaluated against L. monocytogenes LO28, EGD-e and 10403S and further continued with the most resistant strain (10403S) to the combined treatment. For first time, a synergistic effect of liquid smoke and HHP was observed and was further enhanced by freezing prior to HHP. The effect of HHP and liquid smoke, prior to freezing was highest in BHI compared to raw and smoked trout. A major synergistic effect of HHP, liquid smoke and freezing was observed, reaching a 5.48 or 1.93 log CFU/g reduction when smoked or raw trout was used respectively. Furthermore, high injury levels occurred, among treatments reaching up to 55.98%

The Role of Proteasome Beta Subunits in Gastrin-Mediated Transcription of Plasminogen Activator Inhibitor-2 and Regenerating Protein1

The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1(Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role

Perspective on the control of invasive mesquite trees and possible alternative uses

Mesquite trees continue to invade forests and range lands in many countries across the world. The cost to remove these trees is staggering. In Texas, landowners spent $25 million over a 10-year period to clear 300.000 ha of mesquite trees, a fraction of the 22 million ha of Texas land affected by this invasion. Estimates are that the mesquite continues to negatively impact one to two percent of additional land in selected counties each year in Texas. However, the problem is not unique to Texas, but rather to the 44 species of mesquite trees, belonging to the genus Prosopis found in the pea family (Fabaceae), introduced across the southern United States, South Asia, Africa, the Middle East, South America, and the Caribbean. In response, researchers are searching for economically viable uses for harvested trees and seeds to provide an alternative to the high cost of removal. If viable uses for harvested mesquite trees and seeds are found, then sustained pressure will limit and ultimately reduce the negative impact from these invasive trees. One key factor to controlling this invasive species is to find economically and environmentally sustainable uses to help pay the costs of removal or perhaps make removal less necessary. Traditional uses of mesquite are as a building material, as a source of food for both animals and humans and as wood for charcoal. Emerging uses of mesquite are new applications as a biofuel and as a bio-filter medium for water. Moreover, forestry land management of mesquite has adapted to include the tree as a component of hunting lands. New control methodologies and technologies are based on an increased understanding of mesquite growth patterns, using recommended practices that reduce control and eradication costs while improving the efficiency of land management. Previous land management practices have proven that excessive application of herbicides, physical removal of mesquite trees, or human-induced brush fires, if not carefully planned, only worsen mesquite infestations. The growing problem of mesquite land management provides an opportunity for continued research into novel ways to utilize mesquite biomass, of both wood and seed pods

Sweetgum: a new look

Sweetgum (Liquidambar styraciflua L.) is the only species of its genus in the Western hemisphere. The species is a relatively early successional species with wide seed dispersal, fast growth and is considered one of the most adaptable tree species in North America, growing across a wide range of soil types, altitudes, and hydrologic conditions. This species has routinely been considered a lesser desired species by many forest managers trying to grow tree plantations or even in natural stands because the species tends to rapidly invade and dominate a site. However, because of sweetgum’s adaptability, ease of propagation and field planting, and fast growth rate, the tending of sweetgum as a potential crop for improved markets has been reinvigorated. Managing sweetgum also opens the possibility of development of new products and markets that supplement the traditional markets and can produce further value-added products. Increasingly, sweetgum is not viewed with as much antipathy amongst foresters and its potential as valuable resources is being rediscovered