Reorientation of cellulose nanowhiskers in agarose hydrogels under tensile loading

Agarose hydrogels filled with cellulose nanowhiskers were strained in uniaxial stretching under different humidity conditions. The orientation of the cellulose whiskers was examined before and after testing with an X-ray laboratory source and monitored in situ during loading by synchrotron X-ray diffraction. The aim of this approach was to determine the process parameters for reorienting the cellulose nanowhiskers toward a preferential direction. Results show that a controlled drying of the hydrogel is essential to establish interactions between the matrix and the cellulose nanowhiskers which allow for a stress transfer during stretching and thereby promote their alignment. Rewetting of the sample after reorientation of the cellulose nanowhiskers circumvents a critical increase of stress. This improves the extensibility of the hydrogel and is accompanied by a further moderate alignment of the cellulose nanowhiskers. Following this protocol, cellulose nanowhiskers with an initial random distribution can be reoriented toward a preferential direction, creating anisotropic nanocomposites.     

The distribution of the DEK protein in mammalian chromatin

DEK is an abundant and ubiquitous chromatin protein. Here we investigate whether DEK is regularly distributed in the chromatin of human HeLa cells. We show that DEK appears to be excluded from the heterochromatic compartment. However, DEK seems to colocalize with a subfraction of chromatin bearing acetylated histone H4. We examined certain DNA sequences in specifically immunoprecipitated chromatin for four selected human genes. We found that most of the investigated gene sequences were moderately enriched in immunoprecipitated chromatin. In contrast, a promoter-proximal element of the human TOP1 gene was highly enriched in the chromatin immunoprecipitates. This enrichment was lost when cells were treated with alpha-amanitin showing that DEK binds to this particular site only when the TOP1 gene is actively expressed. Our conclusion is that DEK could serve as an architectural protein at the promoter or enhancer sites of a subfraction of human genes.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Isolation of high molecular weight DNA suitable for BAC library construction from woody perennial soft-fruit species

We have developed a novel nuclei extraction method that allows for the extraction of high molecular weight DNA from leaves of woody perennial soft-fruit species that contain high levels of carbohydrates and polyphenolics. The method utilizes a modified buffer system including 4% (w/v) polyvinylpyrrolidone (PVP)-10 and a combination of nylon filters and Percoll gradients to purify nuclei extracts prior to embedding in agarose plugs. The effectiveness of the method was demonstrated on leaves of red raspberry (Rubus idaeus) and blackcurrant (Ribes nigrum), two soft-fruit species that have shown to be recalcitrant to standard genomic DNA extraction methods. Extracted DNA was readily digested by restriction enzymes and, as shown for raspberry, suitable for bacterial artificial chromosome (BAC) library construction.     

Decimal Place Slope,A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of Microbial Species

The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of ¹³C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [¹³C₆]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of ¹³C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful component of qualitative and quantitative proteome studies (1). Incorporation of heavy isotopes can be used to analyze microbial processes such as turnover rates and also to help to establish structure-function relationships within microbial communities. Stable isotope probing (SIP¹) techniques based on DNA-SIP (2) and RNA-SIP (3) have been used for this purpose previously. With the introduction of protein-SIP (4), the need for an accurate alternative method for calculating label incorporation into biomolecules arose. Protein-SIP has several advantages compared with DNA/RNA-SIP, the most important being its capacity to detect dynamic levels of incorporation, whereas only labeled or unlabeled states can be categorized by means of DNA/RNA-SIP because of the need to separate ¹³C-DNA/RNA by density gradient centrifugation. Quantitative analysis of ¹³C incorporation levels is of the utmost importance, especially when unraveling carbon fluxes through either microbial communities or food webs with different trophic levels.In contrast to the incorporation of isotopically labeled amino acids, which is often used in quantitative proteomics (5), metabolic labeling by growth substrates and nutrients (e.g. salts) is often imperfect and makes the processing of mass spectrometry (MS) data difficult. For example, when the incorporation of ¹³C exceeds ∼2 atom %, common database search algorithms fail to identify peptides and proteins. The problem can only be managed successfully if a stable, known degree of ¹³C incorporation can be achieved during the experiment (6). Using a low labeling efficiency of roughly 5 atom %, Huttlin et al. (6) chose the altered envelope chain for calculating the incorporation and simultaneously used the signal intensity for a quantitative comparison with the sample that had a natural abundance of ¹³C. Database approaches for peptide identification can cope only with the natural abundance of carbon isotopes; they fail if the incorporation of ¹³C significantly exceeds the natural isotope abundance or if incorporation patterns occur in unpredictable ways (7).The simplest method for determining the incorporation level is to compare the unlabeled average mass of the monoisotopic peptide with the mass of the labeled protein, as estimated by matrix-assisted laser desorption/ionization or electrospray ionization MS (8, 9). A more advanced approach for determining the isotopic mass distribution of peptides is based on the isotopic distribution of the peaks of a peptide envelope (10, 11). Here, for a given isotopomer, the incorporation efficiency is defined as the percentage of incorporated ¹³C atoms with relation to the total number of carbon atoms with the natural isotope abundance (approximately 1.01 atom % ¹³C). As a reference, the theoretical isotopic distribution of a peptide is calculated based upon an algorithm described elsewhere (12). The isotope distribution of both unlabeled and labeled peptides can subsequently be used to calculate the incorporation level. For this method, an Excel spreadsheet (ProSIPQuant.xls) was developed (4). A similar approach, also based on the calculation of isotopic distributions, has been used in other studies (7). In these studies, however, the identification of the peptides is limited to those that have unlabeled counterparts; in addition, an exact calculation can be hampered by overlapping signals coming from additional peaks with similar masses.In the present study, we describe a new way of determining the isotope incorporation level. Our method makes use of characteristic patterns in the digits after the decimal point of the peptide masses generated by high-accuracy instruments such as the linear ion trap LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). For tryptic peptides, typical regularities in the decimal places of the monoisotopic masses have been observed (13, 14). These observations have been explored in detail for theoretical and experimental data of proteins originating from Helicobacter pylori (15). As a result, a rule called the “half decimal place rule” (HDPR) was defined; it states that the decimal place is nearly half of the first digit for tryptic peptides with masses in the range of 500–1,000 Da. In other words, the exact mass of a peptide is equal to its nominal mass times ∼1.005. Because the difference between ¹²C and ¹³C is slightly greater than 1 Da, exactly 1.0033548378, the decimal places of a tryptic peptide''s mass are shifted in a regular manner by the incorporation level and lead to a significantly increased slope for the digits in the third and fourth place after the decimal point. This shift can be used to estimate the incorporation level of heavy isotopes into the protein. Detecting such shifts requires the highly accurate measurement possible with modern mass spectrometers such as the LTQ-Orbitrap, the Fourier transform ion cyclotron resonance, or the quadrupole time of flight. In this communication, we demonstrate the applicability of this approach using Pseudomonas putida ML2 proteins labeled uniformly via the consumption of [¹³C₆]benzene with five different substrate concentrations (0, 10, 25, 50, and 100 atom % of ¹³C). The ¹³C incorporation was calculated based on several labeled peptides derived from different proteins in one SDS-PAGE band. By these means, we have established a method that allows the determination of ¹³C incorporation into proteins and can be used to assess the metabolic activity of a given species within a mixed community.     

A consistent phylogenetic backbone for the fungi

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.     

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.     

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca²⁺ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF''s profibrotic function in the heart.