Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD⁺ complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD⁺ as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.     

Deficiency of the Promyelocytic Leukemia Protein Fosters Hepatitis C-Associated Hepatocarcinogenesis in Mice

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC. The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase. Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver pathology.     

Direct electrical stimulation of human cortex - the gold standard for mapping brain functions?

Despite its clinical relevance, direct electrical stimulation (DES) of the human brain is surprisingly poorly understood. Although we understand several aspects of electrical stimulation at the cellular level, surface DES evokes a complex summation effect in a large volume of brain tissue, and the effect is difficult to predict as it depends on many local and remote physiological and morphological factors. The complex stimulation effects are reflected in the heterogeneity of behavioural effects that are induced by DES, which range from evocation to inhibition of responses - sometimes even when DES is applied at the same cortical site. Thus, it is a misconception that DES - in contrast to other neuroscience techniques - allows us to draw unequivocal conclusions about the role of stimulated brain areas.     

Clinical Efficacy of Simulated Vitreoretinal Surgery to Prepare Surgeons for the Upcoming Intervention in the Operating Room

Purpose

To evaluate the efficacy of the virtual reality training simulator Eyesi to prepare surgeons for performing pars plana vitrectomies and its potential to predict the surgeons’ performance.

Methods

In a preparation phase, four participating vitreoretinal surgeons performed repeated simulator training with predefined tasks. If a surgeon was assigned to perform a vitrectomy for the management of complex retinal detachment after a surgical break of at least 60 hours it was randomly decided whether a warmup training on the simulator was required (n = 9) or not (n = 12). Performance at the simulator was measured using the built-in scoring metrics. The surgical performance was determined by two blinded observers who analyzed the video-recorded interventions. One of them repeated the analysis to check for intra-observer consistency. The surgical performance of the interventions with and without simulator training was compared. In addition, for the surgeries with simulator training, the simulator performance was compared to the performance in the operating room.

Results

Comparing each surgeon’s performance with and without warmup trainingshowed a significant effect of warmup training onto the final outcome in the operating room. For the surgeries that were preceeded by the warmup procedure, the performance at the simulator was compared with the operating room performance. We found that there is a significant relation. The governing factor of low scores in the simulator were iatrogenic retinal holes, bleedings and lens damage. Surgeons who caused minor damage in the simulation also performed well in the operating room.

Conclusions

Despite the large variation of conditions, the effect of a warmup training as well as a relation between the performance at the simulator and in the operating room was found with statistical significance. Simulator training is able to serve as a warmup to increase the average performance.     

IL-2 / α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment

Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM rejection, as their effect is not sufficiently restricted to Tregs but rather extends to several other lymphocyte populations.     

Prasiolales (Trebouxiophyceae,Chlorophyta) of the Svalbard Archipelago: diversity,biogeography and description of the new genera Prasionella and Prasionema

Species of Prasiolales (Trebouxiophyceae, Chlorophyta) are among the most common terrestrial and freshwater algae in polar regions. Comprehensive molecular studies of this group are available for Antarctica, but not yet for Arctic regions. We examined the diversity of the Prasiolales in the Svalbard Archipelago combining morphological observations of field-collected material, culture studies, molecular data (plastid rbcL and tufA sequences) and literature records. We confirmed the widespread occurrence of Prasiola crispa and P. fluviatilis, species recorded from Spitsbergen since the 19th century. Molecular phylogenetic analyses led to the discovery of two new genera of Prasiolales. Prasionema payeri is morphologically identical to filamentous stages of P. crispa, but represents an early-diverging lineage in the order. Prasionella wendyae is a colonial alga reproducing by aplanospores; its phylogenetic position is among the basal lineages of the order, but it could not be reliably reconstructed due to weak statistical support. The inclusion of P. wendyae in the prasiolalean phylogeny determined the paraphyly of Rosenvingiella, requiring the establishment of the new genus Rosenvingiellopsis for R. constricta. A poorly known species described from Spitsbergen, Ulothrix discifera, is transferred here to Rosenvingiella. Whereas some species of Prasiolales have bipolar distribution (P. crispa), others appear to be restricted to one or other of the poles. Our results suggest that polar regions are still a major repository of unknown algal diversity and highlight the importance of continued field surveys and the use of molecular data.     

Tyrosine kinase and cooperative TGFbeta signaling in the reproductive organs of Schistosoma mansoni

Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. The establishment of drug targets requires knowledge about the molecular mechanisms that regulate the development of the female reproductive organs, which include vitellarium and ovary. This review summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, especially cytoplasmatic tyrosine kinases of the Src class seem to play dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGFbeta signaling in the production of vitellocytes. Finally, we take advantage from the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.     

New Streptococcus pneumoniae clones in deceased wild chimpanzees

In wild chimpanzees in the Ta? National Park, C?te d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of Streptococcus pneumoniae were identified in deceased apes on the basis of multilocus sequence typing analysis and ply, lytA, and pbp2x sequences. The findings suggest that virulent S. pneumoniae occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans.     

Fucoidans and fucoidanases—focus on techniques for molecular structure elucidation and modification of marine polysaccharides

The research field of fucoidans (sulphated polysaccharides from algae) and fucoidanases was strongly developing in recent years. Several different fucoidans and a few fucoidan-degrading enzymes were isolated and characterised. A high potential is seen in the medical exploitation of the fucoidans and its degradation products. This review gives an overview about the research of the last 5 years concerning fucoidan characterisation and application as well as enzyme detection, characterisation and production.