Autoradiographic and electron microscopic studies of minced cardiac muscle regeneration in the adult newt, notophthalmus viridescens.

The regenerative response of minced cardiac muscle grafts in the adult newt was studied using autoradiography and electron microscopy. One-sixteenth to one-eighth of the newt ventricle was amputated, minced, and returned to the wounded ventricle. At five days after grafting, no reorganization of graft msucle pieces was apparent and there was degeneration of much of the muscle graft. Another, smaller population of 5-day myocytes had euchromatic nuclei and intact sarcolemmae. In 10- and 16-day grafts, continuity between ventricular and graft lumina was established and coalescence of graft pieces was apparent. Ultrastructurally, 10- and 16-day graft myocytes appeared to have fewer myofibrillae and increased amounts of rough endoplasmic reticulum, polyribosomes, Golig complexes, and dense bodies when compared to uninjured ventricular myocytes. The peak of proliferative activity of graft cells was observed at 16 days. Electron microscopic autoradiography revealed breadkdown of myofibrillar structure in labeled myocytes, whereas in myocytes in the later stages of mitosis only scattered myofilaments and no Z bands were present. By 30 days, grafts appeared as an integrated structure composed primarily of cardiac muscle. Myocytes of 30-day grafts were observed in various stages of myofibrillogenesis and contained numberous 10-nm filaments. Seventy-day graft mycoytes had numberous well organized myofibrillae and intercellular junctions similar to those seen in uninjured ventricular myocytes.     

Inclusion of phytosociological data in an index of vegetation fire danger: application and mapping on the Karst area around Trieste (Italy)

In this paper we propose a method to produce maps of fire danger index (FDI). The index includes in the formula a vegetation pyrogenic potential index (VPPI) based on floristic phytosociological data. The map of FDI is produced by integrating available phytosociological maps of vegetation, geomorphology and climate, using the Geographic Information System technology. The method is applied to an area of the coastal Classical Karst (NE-Italy). While fire risk maps based on the incidence of previous fires are useful for facilitating emergency operations, e.g. the allocation of fire fighting resources, we conclude that the proposed index offers an effective tool to plan actions for fire prevention.     

Identification of the ubiquinone-binding domain in the disulfide catalyst disulfide bond protein B.

Disulfide bond (Dsb) formation is catalyzed in the periplasm of prokaryotes by the Dsb proteins. DsbB, a key enzyme in this process, generates disulfides de novo by using the oxidizing power of quinones. To explore the mechanism of this newly described enzymatic activity, we decided to study the ubiquinone-protein interaction and identify the ubiquinone-binding domain in DsbB by cross-linking to photoactivatable quinone analogues. When purified Escherichia coli DsbB was incubated with an azidoubiquinone derivative, 3-azido-2-methyl-5-[(3)H]methoxy-6-decyl-1,4-benzoquinone ([(3)H]azido-Q), and illuminated with long wavelength UV light, the decrease in enzymatic activity correlated with the amount of 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q) incorporated into the protein. One azido-Q-linked peptide with a retention time of 33.5 min was obtained by high performance liquid chromatography of the V8 digest of [(3)H]azido-Q-labeled DsbB. This peptide has a partial NH(2)-terminal amino acid sequence of NH(2)-HTMLQLY corresponding to residues 91-97. This sequence occurs in the second periplasmic domain of the inner membrane protein DsbB in a loop connecting transmembrane helices 3 and 4. We propose that the quinone-binding site is within or very near to this sequence.     

The temperature acclimation potential of tropical bryophytes

Bryophyte biomass and diversity in tropical moist forests decrease dramatically from higher altitudes towards the lowlands. High respiratory carbon losses at high temperatures may partly explain this pattern, if montane species are unable to acclimatise their metabolic rates to lowland temperatures. We transplanted ten bryophyte species from two altitudes (1200 and 500 m a.s.l.) to lower (warmer) altitudes (500 m and sea level) in Panama. We studied short‐term temperature acclimation of CO₂ exchange for 2.5 months, and survival and growth for 21 months following transplantation. Short‐term acclimation did not occur, and on a longer time scale mortality was highest and growth lowest in the transplanted samples. A few transplanted samples of most species, however, survived the whole experiment and finished with growth rates similar to controls. This recovery of growth rate suggests temperature acclimation, in spite of no measurable metabolic changes in smaller random samples. This acclimation even compensated for shorter periods of CO₂ uptake due to more rapid drying. Nevertheless, these species are not abundant in lowland forests, perhaps due to dispersal or establishment limitation. The apparent heterogeneity of the acclimation potential within species may allow populations to adapt locally and avoid being forced uphill under climatic warming.     

Key parameters controlling stiffness variability within trees: a multiscale experimental–numerical approach

Microstructural properties of wood vary considerably within a tree. Knowledge of these properties and a better understanding of their relationship to the macroscopic mechanical performance of wood are crucial to optimize the yield and economic value of forest stocks. This holds particularly for the end-use requirements in engineering applications. In this study the microstructure–stiffness relationships of Scots pine are examined with a focus on the effects of the microstructural variability on the elastic properties of wood at different length scales. For this purpose, we have augmented microstructural data acquired using SilviScan-3? (namely wood density, cell dimensions, earlywood and latewood proportion, microfibril angle) with local measurements of these quantities and of the chemical composition derived from wide-angle X-ray scattering, light microscopy, and thermogravimetric analysis, respectively. The stiffness properties were determined by means of ultrasonic tests at the clear wood scale and by means of nanoindentation at the cell wall scale. In addition, micro-mechanical modeling was applied to assess the causal relations between structural and mechanical properties and to complement the experimental investigations. Typical variability profiles of microstructural and mechanical properties are shown from pith to bark, across a single growth ring and from earlywood to latewood. The clear increase of the longitudinal stiffness as well as the rather constant transverse stiffness from pith to bark could be explained by the variation in microfibril angle and wood density over the entire radial distance. The dependence of local cell wall stiffness on the local microfibril angle was also demonstrated. However, the local properties did not necessarily follow the trends observed at the macroscopic scale and exhibited only a weak relationship with the macroscopic mechanical properties. While the relationship between silvicultural practice and wood microstructure remains to be modeled using statistical techniques, the influence of microstructural properties on the macroscopic mechanical behavior of wood can now be described by a physical model. The knowledge gained by these investigations and the availability of a new micromechanical model, which allows transferring these findings to non-tested material, will be valuable for wood quality assessment and optimization in timber engineering.     

Brain serotonin deficiency leads to social communication deficits in mice

A deficit in brain serotonin is thought to be associated with deteriorated stress coping behaviour, affective disorders and exaggerated violence. We challenged this hypothesis in mice with a brain-specific serotonin depletion caused by a tryptophan hydroxylase 2 (TPH2) deficiency. We tested TPH2-deficient (Tph2^−/–) animals in two social situations. As juveniles, Tph2^−/− mice displayed reduced social contacts, whereas ultrasonic vocalizations (USVs) were unchanged within same-sex same-genotype pairings. Interestingly, juvenile females vocalized more than males across genotypes. Sexually naive adult males were exposed to fresh male or female urine, followed by an interaction with a conspecific, and re-exposed to urine. Although Tph2^−/− mice showed normal sexual preference, they were hyper-aggressive towards their interaction partners and did not vocalize in response to sexual cues. These results highlight that central serotonin is essential for prosocial behaviour, especially USV production in adulthood, but not for sexual preference.     

Stability-enhanced Hot-melt Extruded Amorphous Solid Dispersions via Combinations of Soluplus? and HPMCAS-HF

The aim of this study was to evaluate a novel combination of Soluplus® and hypromellose acetate succinate (HPMCAS-HF) polymers for solubility enhancement as well as enhanced physicochemical stability of the produced amorphous solid dispersions. This was accomplished by converting the poorly water-soluble crystalline form of carbamazepine into a more soluble amorphous form within the polymeric blends. Carbamazepine (CBZ), a Biopharmaceutics Classification System class II active pharmaceutical ingredient (API) with multiple polymorphs, was utilized as a model drug. Hot-melt extrusion (HME) processing was used to prepare solid dispersions utilizing blends of polymers. Drug loading showed a significant effect on the dissolution rate of CBZ in all of the tested ratios of Soluplus® and HPMCAS-HF. CBZ was completely miscible in the polymeric blends of Soluplus® and HPMCAS-HF up to 40% drug loading. The extrudates were characterized by differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and dissolution studies. DSC and XRD data confirmed the formation of amorphous solid dispersions of CBZ in the polymeric blends of Soluplus® and HPMCAS-HF. Drug loading and release of CBZ was increased with Soluplus® (when used as the primary matrix polymer) when formulations contained Soluplus® with 7–21% (w/w) HPMCAS-HF. In addition, this blend of polymers was found to be physically and chemically stable at 40°C, 75% RH over 12 months without any dissolution rate changes.KEY WORDS: carbamazepine, hot-melt extrusion, HPMCAS-HF, Soluplus®, stability     

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)¹ are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue.