Delayed Contrast Extravasation MRI for Depicting Tumor and Non-Tumoral Tissues in Primary and Metastatic Brain Tumors

The current standard of care for newly diagnosed glioblastoma multiforme (GBM) is resection followed by radiotherapy with concomitant and adjuvant temozolomide. Recent studies suggest that nearly half of the patients with early radiological deterioration post treatment do not suffer from tumor recurrence but from pseudoprogression. Similarly, a significant number of patients with brain metastases suffer from radiation necrosis following radiation treatments. Conventional MRI is currently unable to differentiate tumor progression from treatment-induced effects. The ability to clearly differentiate tumor from non-tumoral tissues is crucial for appropriate patient management. Ten patients with primary brain tumors and 10 patients with brain metastases were scanned by delayed contrast extravasation MRI prior to surgery. Enhancement subtraction maps calculated from high resolution MR images acquired up to 75 min after contrast administration were used for obtaining stereotactic biopsies. Histological assessment was then compared with the pre-surgical calculated maps. In addition, the application of our maps for prediction of progression was studied in a small cohort of 13 newly diagnosed GBM patients undergoing standard chemoradiation and followed up to 19.7 months post therapy. The maps showed two primary enhancement populations: the slow population where contrast clearance from the tissue was slower than contrast accumulation and the fast population where clearance was faster than accumulation. Comparison with histology confirmed the fast population to consist of morphologically active tumor and the slow population to consist of non-tumoral tissues. Our maps demonstrated significant correlation with perfusion-weighted MR data acquired simultaneously, although contradicting examples were shown. Preliminary results suggest that early changes in the fast volumes may serve as a predictor for time to progression. These preliminary results suggest that our high resolution MRI-based delayed enhancement subtraction maps may be applied for clear depiction of tumor and non-tumoral tissues in patients with primary brain tumors and patients with brain metastases.     

Vascular supply of the anterior interventricular epicardial nerves and ventricular Purkinje fibers in the porcine hearts

The aim of this study was to perform a pilot histological and quantitative analysis of the blood vessels accompanying the epicardial nerves (vasa nervorum) in the porcine hearts. Twenty healthy porcine hearts were used in this study. The blood vessels were analyzed by light microscopy using four different staining techniques in transverse sections taken from the upper, middle, and lower segments of the anterior part of the interventricular region and the adjacent parts of the right and left ventricles containing epicardial nerves and the endocardial peripheral parts of the Purkinje fibers. In total, 317 epicardial nerves were detected. The vasa nervorum were present in 75.7% of these nerves. The vasa nervorum resembled arterioles and postcapillary and collecting venules. One hundred and forty nine epicardial nerves were perivascular, located in the adventitia of the anterior interventricular artery and vein. The remaining 168 nerves ran freely through the epicardial interstitium. The presence of the vasa nervorum was not related to topographical location or nerve diameter. Additionally, from a total of 33 analyzed ventricular complexes of Purkinje fibers small blood vessels located in their proximity were identified in only two cases. It can be concluded that the majority of the anterior epicardial nerves of porcine heart possess well-developed vasa nervorum. In contrast, similar blood vessels are rarely present in the vicinity of the Purkinje fibers. The data obtained contribute to a better understanding of the nutrition of the cardiac nerves.     

Range expansion of the small spruce bark beetle Ips amitinus: a newcomer in northern Europe

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Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells

In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or genetically manipulated mouse lines. These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.     

Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin.     

Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b₆f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.Desiccation tolerance, the ability to survive absolute water contents down to approximately 0.1 g water g⁻¹ dry weight, is a trait found in some bacteria, algae, fungi, as well as animals and plants. In the plant kingdom, desiccation tolerance is common in ferns, mosses, and most seeds and pollen of flowering plants (angiosperms). Resurrection plants, a diverse group of approximately 300 angiosperm species, possess this trait also in their vegetative tissues. These plants are able to withstand prolonged periods of dehydration and to recover within hours to a few days once water is available. A major and interesting aspect in the study of desiccation tolerance in resurrection plants is how they protect themselves against oxidative damage during dehydration, which is often accompanied by conditions of high irradiance (for review, see Bartels and Hussain, 2011; Farrant and Moore, 2011; Morse et al., 2011).A decrease in water content quickly results in lowered leaf stomatal conductance and, consequently, decreased uptake of CO₂. This hinders and ultimately blocks the Calvin cycle. The light-driven reactions, however, typically continue well after the onset of water deficiency, with intact chlorophyll-protein complexes absorbing light energy. The imbalance between the light reactions and the downward biochemical pathways results in a lack of electron sinks and in the system becoming overenergized. This, in turn, leads to enhanced generation of reactive oxygen species (ROS), which inflict damage onto photosynthetic components as well as onto other chloroplast and cellular constituents. At times, the damage may be severe and lead to irreversible impairment and finally plant death (Dinakar et al., 2012).Resurrection plants minimize such potential ROS damage by shutting down photosynthesis during early stages of dehydration (Farrant, 2000; Farrant et al., 2007). There are two mechanisms whereby this is achieved. In poikilochlorophyllous resurrection plants, chlorophyll, along with photosynthetic protein complexes, are degraded, and thylakoids, the membranes that host the photosynthetic pigment-protein complexes, are dismantled. This straightforward mechanism prevents the formation of ROS, yet it comes at the cost of resynthesizing photosynthetic components de novo upon rehydration. On the other hand, homoiochlorophyllous species retain most of their photosynthetic complement and so must rely on other means to protect themselves from oxidative damage in the desiccated state. Some of these, such as leaf folding or curling, which minimize the exposure of inner leaves and/or of adaxial (upper) leaf surfaces to the light, and the accumulation of anthocyanins in leaf surfaces, which act as sunscreens, and the presence of reflective hairs and waxy cuticles, reduce the overall absorption of radiation and thus protect against photodamage (Sherwin and Farrant, 1998; Farrant, 2000; Bartels and Hussain, 2011; Morse et al., 2011). ROS that are generated are dealt with by antioxidants, ROS scavengers, and in some cases also by anthocyanins and other polyphenols (Moore et al., 2005; Kytridis and Manetas, 2006; Farrant et al., 2007). Nevertheless, all of these mechanisms are insufficient to completely prevent and/or detoxify all ROS that are formed, necessitating additional means to prevent or deal with possible damage that ROS may inflict during dehydration and while desiccated (Dinakar et al., 2012).The major photoprotective mechanism in plants and algae is nonphotochemical quenching (NPQ), in which excess light energy absorbed at the antennae of PSII is dissipated as heat. NPQ has been shown to be active in desiccation-tolerant bryophytes and pteridiophytes (Eickmeier et al., 1993; Oliver, 1996), in homoiochlorophyllous angiosperms (Alamillo and Bartels, 2001; Georgieva et al., 2009; Dinakar and Bartels, 2012; Huang et al., 2012), and during the initial stages of drying in poikilochlorophyllous angiosperms (Beckett et al., 2012). Photoinhibition, when damage to PSII (mainly to its D1 subunit) exceeds the repair capacity, typically under conditions of light stress, is also observed in homoiochlorophyllous resurrection plants (e.g. Georgieva and Maslenkova, 2006). Other ways to avoid ROS-induced damage include the rerouting of reducing equivalents to alternative electron sinks, such as the water-water cycle and/or photorespiration, as well as structural rearrangements of PSII and light-harvesting antenna (LHCII) complexes into energy-dissipating states (for review, see Dekker and Boekema, 2005; Yamamoto et al., 2014). These latter processes, in particular the ones pertaining to possible changes in PSII-LHCII macrostructure, have not yet been characterized in homoiochlorophyllous resurrection plants.To gain insight into the ways homoiochlorophyllous resurrection plants cope with dehydration while retaining most of their photosynthetic apparatus, we combined microscopic, spectroscopic, and biochemical approaches. Investigation of the supramolecular organization of photosynthetic complexes was carried out using cryoscanning electron microscopy (cryo-SEM) of high-pressure frozen, freeze-fractured leaf samples; to our knowledge, this combination of procedures has not been utilized previously to investigate thylakoid membranes within plant tissues.The studies reveal that during dehydration, the density of PSII in grana membranes gradually decreases. Notably, in the dehydrated state, in which photosynthetic activity is halted, PSII complexes are also observed to be arranged into rows and two-dimensional arrays. These arrangements are proposed to represent quenched PSII complexes that likely minimize the generation of ROS during desiccation. Furthermore, we observe inverted hexagonal (H_II) phases in this dry state, and these two structural rearrangements are correlated with the massive accumulation of Suc. Biochemical studies of thylakoid membrane fractions support the finding that the relative level of PSII proteins decreases during dehydration. These analyses also reveal that the level of the cytochrome f subunit of the cytochrome b₆f complex decreases quite dramatically and early during dehydration. This provides evidence for an additional level of regulation that inhibits/shuts down the photosynthetic light reactions during desiccation.     

Xylosandrus germanus in Central Europe: Spread into and within the Czech Republic

Invasive organisms represent great threats to ecosystems and great challenges to forest management. In Europe, the black timber bark beetle (Xylosandrus germanus) is an invasive secondary pest that mostly attacks the logs of felled trees. We showed the invasion history for Europe and using many local surveys, we summarize the current distribution and other available information on X. germanus in the Czech Republic. We report that this species is distributed from the lowlands to the mountains in the Czech Republic; it is widespread in the eastern half of the country, where it is more abundant in the warmer south and southeast areas than in the cooler areas. Most (78%) of the known localities are at elevation below 400 m a.s.l. Although an ice storm greatly increased X. germanus abundance near the border with Austria, its high abundance did not result in damage to standing trees. Presence of X. germanus in the Czech Republic for over 10 years has not led to heavy tree infestation.