Identification of an atypical interaction site in the BTB domain of the MYC-interacting zinc-finger protein 1

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Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3’ ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.     

Reductive Biotransformation of Benzaldehyde Derivatives by Baker's Yeast in Non-Conventional Media: Effect of Substrate Hydrophobicity on the Biocatalytic Reaction

The reduction of six derivatives of benzaldehyde, α,α,α-trifluorotolualdehyde, nitrobenzaldehyde, anisaldehyde, fluorobenzaldehyde, tolualdehyde, chlorobenzaldehyde, each of which was substituted in the ortho-, meta- and para-positions, was investigated in whole cell yeast biotransformations conducted in non-conventional media. Correlation between hydrophobic (π) and electronic (σ) parameters of the substituent of the substrate and biocatalytic activity in organic solvent media (hexane containing 2% v/v water) was evaluated. While catalytic activity in general decreased as the substituent hydrophobicity (π) increased, the trend was more pronounced for ortho- substituents compared to meta- and para-. When the electronic parameters (σ) of the meta- and para- substituents were correlated with the catalytic activity, the opposite was observed, namely the catalytic activity increased as the electronic parameter of the substituent increased. This observation was similar for meta- and para- substituents.

Preliminary studies on the relationships between solvent and substrate polarity on the one hand and catalytic activity on the other are also presented and discussed.     

Validation of density–elasticity relationships for finite element modeling of human pelvic bone by modal analysis

In total hip arthroplasty and particularly in revision surgery, computer assisted pre-operative prediction of the best possible anchorage strategy for implant fixation would be a great help to the surgeon. Computer simulation relies on validated numerical models. In the current study, three density–elasticity relationships (No. 1–3) from the literature for inhomogeneous material parameter assignment from CT data in automated finite element (FE) modeling of long bones were evaluated for their suitability for FE modeling of human pelvic bone. Numerical modal analysis was conducted on 10 FE models of hemipelvic bone specimens and compared to the gold standard provided by experimental modal analysis results from a previous in-vitro study on the same specimens. Overall, calculated resonance frequencies came out lower than measured values. Magnitude of mean relative deviation of numerical resonance frequencies with regard to measured values is lowest for the density–elasticity relationship No. 3 (−15.9%) and considerably higher for both density–elasticity relationships No. 1 (−41.1%) and No. 2 (−45.0%). Mean MAC values over all specimens amount to 77.8% (No. 1), 78.5% (No. 2), and 83.0% (No. 3). MAC results show, that mode shapes are only slightly influenced by material distribution. Calculated resonance frequencies are generally lower than measured values, which indicates, that numerical models lack stiffness. Even when using the best suited (No. 3) out of three investigated density–elasticity relationships, in FE modeling of pelvic bone a considerable underestimation of model stiffness has to be taken into account.     

Seeing is believing: Dynamic changes in renal epithelial autophagy during injury and repair

Ischemic injury to the kidneys is a prevalent clinical problem, contributing importantly to chronic kidney disease. Yet, underlying molecular mechanisms are elusive. To address the possible role of autophagy, we engineered a novel strain of mice harboring a ubiquitously expressed CAG-RFP-EGFP-LC3 transgene. Using this tool, we examined the post-ischemic kidney and detailed the dynamics of renal tubular epithelial autophagy. In addition, we defined the role of MTOR in the resolution of autophagy during epithelial survival and kidney repair.     

Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.     

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.     

Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.