Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4⁺ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4⁺CD25⁺FoxP3⁺ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.     

Ecosystem state shifts during long‐term development of an Amazonian peatland

The most carbon (C)‐dense ecosystems of Amazonia are areas characterized by the presence of peatlands. However, Amazonian peatland ecosystems are poorly understood and are threatened by human activities. Here, we present an investigation into long‐term ecohydrological controls on C accumulation in an Amazonian peat dome. This site is the oldest peatland yet discovered in Amazonia (peat initiation ca. 8.9 ka BP), and developed in three stages: (i) peat initiated in an abandoned river channel with open water and aquatic plants; (ii) inundated forest swamp; and (iii) raised peat dome (since ca. 3.9 ka BP). Local burning occurred at least three times in the past 4,500 years. Two phases of particularly rapid C accumulation (ca. 6.6–6.1 and ca. 4.9–3.9 ka BP), potentially resulting from increased net primary productivity, were seemingly driven by drier conditions associated with widespread drought events. The association of drought phases with major ecosystem state shifts (open water wetland–forest swamp–peat dome) suggests a potential climatic control on the developmental trajectory of this tropical peatland. A third drought phase centred on ca. 1.8–1.1 ka BP led to markedly reduced C accumulation and potentially a hiatus during the peat dome stage. Our results suggest that future droughts may lead to phases of rapid C accumulation in some inundated tropical peat swamps, although this can lead ultimately to a shift to ombrotrophy and a subsequent return to slower C accumulation. Conversely, in ombrotrophic peat domes, droughts may lead to reduced C accumulation or even net loss of peat. Increased surface wetness at our site in recent decades may reflect a shift towards a wetter climate in western Amazonia. Amazonian peatlands represent important carbon stores and habitats, and are important archives of past climatic and ecological information. They should form key foci for conservation efforts.     

Rock glaciers in crystalline catchments: Hidden permafrost‐related threats to alpine headwater lakes

A global warming‐induced transition from glacial to periglacial processes has been identified in mountainous regions around the world. Degrading permafrost in pristine periglacial environments can produce acid rock drainage (ARD) and cause severe ecological damage in areas underlain by sulfide‐bearing bedrock. Limnological and paleolimnological approaches were used to assess and compare ARDs generated by rock glaciers, a typical landform of the mountain permafrost domain, and their effects on alpine headwater lakes with similar morphometric features and underlying bedrock geology, but characterized by different intensities of frost action in their catchments during the year. We argue that ARD and its effects on lakes are more severe in the alpine periglacial belt with mean annual air temperatures (MAAT) between ?2°C and +3°C, where groundwater persists in the liquid phase for most of the year, in contrast to ARD in the periglacial belt where frost action dominates (MAAT < ?2°C). The findings clearly suggest that the ambient air temperature is an important factor affecting the ARD production in alpine periglacial environments. Applying the paleoecological analysis of morphological abnormalities in chironomids through the past millennium, we tested and rejected the hypothesis that unfavorable conditions for aquatic life in the ARD‐stressed lakes are largely related to the temperature increase over recent decades, responsible for the enhanced release of ARD contaminants. Our results indicate that the ARDs generated in the catchments are of a long‐lasting nature and the frequency of chironomid morphological deformities was significantly higher during the Little Ice Age (LIA) than during pre‐ or post‐LIA periods, suggesting that lower water temperatures may increase the adverse impacts of ARD on aquatic invertebrates. This highlights that temperature‐mediated modulations of the metabolism and life cycle of aquatic organisms should be considered when reconstructing long‐term trends in the ecotoxicological state of lakes.     

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.     

The Essential Role of Mass Spectrometry in Characterizing Protein Structure: Mapping Posttranslational Modifications

Over the last few years we have developed mass spectrometry-based approaches for selective identification of a variety of posttranslational modifications, and for sequencing the modified peptides. These methods do not involve radiolabeling or derivatization. Instead, modification-specific fragment ions are produced by collision-induced dissociation (CID) during analysis of peptides by ESMS. The formation and detection of these marker ions on-the-fly during the LC-ESMS analysis of a protein digest is a powerful technique for identifying posttranslationally modified peptides. Using the marker ion strategy in an orthogonal fashion, a precursor ion scan can detect peptides which give rise to a diagnostic fragment ion, even in an unfractionated protein digest. Once the modified peptide has been located, the appropriate precursor ion can be sequenced by tandem MS. The utility and interplay of this approach to mapping PTM is illustrated with examples that involve protein glycosylation and phosphorylation.     

Conformational analysis of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-α-D-galactopyranoside by MM and Langevin dynamics simulations

The conformational space of methyl 6-O-[(R)- and (S)- 1-carboxyethyl]-α-D-galactopyranoside has been investigated. A grid search employing energy minimization at each grid point over the three major degrees of freedom, namely φ, ψ and ω, identified low energy regions. The R-isomer shows five low energy conformers within ca. 1 kcal mol−1 of the global energy minimum. The S-isomer has two conformers within a few tenths of a kcal mol−1 of the global energy minimum. Langevin dynamics simulations have been have been performed at 300 K for 30 ns of each isomer. The φ dihedral angle has as its major conformer (g−1) for the R-isomer whereas it is the (g+) conformer for the S-isomer. For the ψ dihedral angle the (t) conformer has the highest population for both isomers. The dihedral angle ω has the (g+) conformer most highly populated, both for the R- and S-isomer. The above five and two conformational states for the R- and S-isomers, respectively, make up 90% in each case of the populated states during the Langevin dynamics (LD) simulations. Rate constants for the ω dihedral angle have been calculated based on a number correlation function. Three bond homo- and heteronuclear, i.e. proton and carbon-13, coupling constants have been calculated from the dynamics trajectories for comparison to experimental values. The heteronuclear coupling constant H2′,C6 has been measured for the S-isomer and found to be 3.3 Hz. The J value calculated from the LD simulations, namely 2.6 Hz, is in fair agreement with experiment. A comparison to the X-ray structure of the R-isomer shows that the conformation of the crystalline compound occupies the low energy region most highly populated as a single R-conformer (30%) during the LD simulations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sympathetic nerve regulation of cochlear blood flow during increases in blood pressure in humans

The purpose of this work was to show that regulation of the blood flow to the cochlea by the sympathetic nervous system occurs in humans at the level of the cochlear microcirculation during increases in blood pressure and that its involvement depends on the pressure level. Eight anaesthetized patients undergoing tympanoplasty for hearing disease took part in a pharmacological protocol of stimulation and inhibition of the autonomic nervous system (ANS) to provide variations in systolic blood pressure (BP_S) and cochlear blood flow (CBF). The CBF was measured by laser-Doppler flowmetry. Changes in autonomic nerve activity were brought about by changes in baroreceptor activity (BR) initiated by the injection of an α adrenergic agent before and after sympathetic and parasympathetic blockade. The CBF variations (δCBF) were plotted against BP_S increases at each stage of the ANS inhibition. The BR diminished significantly after α blockade, after α and β blockade, and after α and β blockade and atropine, by 50% (P < 0.01), 29% (P < 0.05), and 95% (P < 0.001) respectively. The BP_S increased significantly (P < 0.01) by 36 (SD 9)%, 47 (SD 1)%, and 67 (SD 16)% respectively. The CBF response to an increase in BP_S exhibited two opposing variations in the patients: CBF decreased significantly in one group, and increased significantly in the other group. In both groups, δCBF decrease and δCBF increase, respectively, were significant after ANS blockade; even so the decrease and increase, respectively, levelled off at BP_S around 160 mmHg before ANS blockade. For BP_S below 160 mmHg, correlations between δCBF and BP_S were significant before inhibition and after inhibition of ANS. For BP_S above 160 mmHg, BP_S and δCBF were not correlated before inhibition of ANS, and were significantly correlated after inhibition of ANS. For BP_S below 160 mmHg, CBF response to the BP_S increase was the same before and after ANS blockade, i.e. ANS control did not predominate; even so, for BP_S above 160 mmHg, the CBF response to BP_S increase was different before and after ANS blockade: CBF varied significantly after ANS blockade as it varied for BP_S below 160 mmHg, while it remained constant before ANS blockade that elicited ANS control of CBF. In conclusion, sympathetic nerve regulation via its vasomotor tone at the level of cochlear microcirculation occurred markedly when the blood pressure was above 160 mmHg; the autonomic nervous system would appear to control the cochlear blood flow against large variations in blood flow in response to hypertensive phenomena. Accepted: 7 October 1996     

Ascospore septation inDiploschistes (Thelotremataceae, lichenizedAscomycota) and the taxonomic significance of macro- and microcephalic ascospore types

The ascospore septation in 13 species ofDiploschistes was examined. Microcephalic, macrocephalic, as well as irregular ascospore septation were found. In some species and even specimens of some species, different types of spore septation were detected. The taxonomic significance of the character of ascospore septation sequence is questioned, and the distinction of the ordersGraphidales andOstropales, which was primarily based on this character, is rejected.This paper is dedicated to Prof. DrRolf Santesson (Uppsala) on the occasion of his 80th birthday.     

Seedling recruitment in the invasive clonal shrub, Mahonia aquifolium Pursh (Nutt.)

The clonal shrub, Mahonia aquifolium, is an aggressive invader of some forests in central Germany. We analysed the importance of seedling recruitment for the local dynamics of these invasive populations. In contrast to many other clonal plants, repeated seedling recruitment takes place in M. aquifolium, contributing to the colonization of available space within populations. Thus, sexual reproduction is not only important for dispersal to new sites but also for the local invasion␣process. In situations with grass competition (Calamagrostis epigejos) the number of successful recruits is reduced, and clonal growth is the predominant mode of reproduction. Since seedling recruitment results in an increasing number of genets as well as ramets, the investigated populations are still below their carrying capacities. Seedling densities are higher beneath the canopy of adults than outside adult patches, and increase with increasing cover of adult ramets. The spatially clumped pattern of seedling emergence results in density-dependent mortality especially in the most crowded seedling clusters. However, adult neighbourhood imposes a much higher mortality. Therefore, we expect that the importance of seedling recruitment for the dynamics of local M. aquifolium populations will decline as the invasion process continues. Received: 15 January 1996 / Accepted: 28 September 1996