Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41-0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67-1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

Impaired top-down modulation of saccadic latencies in patients with schizophrenia but not in first-degree relatives

Impaired eye movements have a long history in schizophrenia research and meet the criteria of a reliable biomarker. However, the effects of cognitive load and task difficulty on saccadic latencies (SL) are less understood. Recent studies showed that SL are strongly task dependent: SL are decreased in tasks with higher cognitive demand, and increased in tasks with lower cognitive demand. The present study investigates SL modulation in patients with schizophrenia and their first-degree relatives. A group of 13 patients suffering from ICD-10 schizophrenia, 10 first-degree relatives, and 24 control subjects performed two different types of visual tasks: a color task and a Landolt ring orientation task. We used video-based oculography to measure SL. We found that patients exhibited a similar unspecific SL pattern in the two different tasks, whereas controls and relatives exhibited 20–26% shorter average latencies in the orientation task (higher cognitive demand) compared to the color task (lower cognitive demand). Also, classification performance using support vector machines suggests that relatives should be assigned to the healthy controls and not to the patient group. Therefore, visual processing of different content does not modulate SL in patients with schizophrenia, but modulates SL in the relatives and healthy controls. The results reflect a specific oculomotor attentional dysfunction in patients with schizophrenia that is a potential state marker, possibly caused by impaired top-down disinhibition of the superior colliculus by frontal/prefrontal areas such as the frontal eye fields.

Complementary Medicine in Nursing Homes - Results of a Mixed Methods Pilot Study

Background ‘Kneipp Therapy’ (KT) is a form of Complementary and Alternative Medicine (CAM) that includes a combination of hydrotherapy, herbal medicine, mind-body medicine, physical activities, and healthy eating. Since 2007, some nursing homes for older adults in Germany began to integrate CAM in the form of KT in care. The study investigated how KT is used in daily routine care and explored the health status of residents and caregivers involved in KT. Methods We performed a cross-sectional pilot study with a mixed methods approach that collected both quantitative and qualitative data in four German nursing homes in 2011. Assessments in the quantitative component included the Quality of Life in Dementia (QUALIDEM), the Short Form 12 Health Survey (SF-12), the Barthel-Index for residents and the Work Ability Index (WAI) and SF-12 for caregivers. The qualitative component addressed the residents’ and caregivers’ subjectively experienced changes after integration of KT. It was conceptualized as an ethnographic rapid appraisal by conducting participant observation and semi-structured interviews in two of the four nursing homes. Results The quantitative component included 64 residents (53 female, 83.2 ± 8.1 years (mean and SD)) and 29 caregivers (all female, 42.0 ± 11.7 years). Residents were multimorbid (8 ± 3 diagnoses), and activities of daily living were restricted (Barthel-Index 60.6 ± 24.4). The caregivers’ results indicated good work ability (WAI 37.4 ± 5.1), health related quality of life was superior to the German sample (SF-12 physical CSS 49.2 ± 8.0; mental CSS 54.1 ± 6.6). Among both caregivers and residents, 89% considered KT to be positive for well-being. The qualitative analysis showed that caregivers perceived emotional and functional benefits from more content and calmer residents, a larger variety in basic care practices, and a more self-determined scope of action. Residents reported gains in attention and caring, and recognition of their lay knowledge. Conclusion Residents showed typical characteristics of nursing home inhabitants. Caregivers demonstrated good work ability. Both reported to have benefits from KT. The results provide a good basis for future projects, e.g. controlled studies to evaluate the effects of CAM in nursing homes.

Honey Bee Apis mellifera Parasites in the Absence of Nosema ceranae Fungi and Varroa destructor Mites

Few areas of the world have western honey bee (Apis mellifera) colonies that are free of invasive parasites Nosema ceranae (fungi) and Varroa destructor (mites). Particularly detrimental is V. destructor; in addition to feeding on host haemolymph, these mites are important vectors of several viruses that are further implicated as contributors to honey bee mortality around the world. Thus, the biogeography and attendant consequences of viral communities in the absence of V. destructor are of significant interest. The island of Newfoundland, Province of Newfoundland and Labrador, Canada, is free of V. destructor; the absence of N. ceranae has not been confirmed. Of 55 Newfoundland colonies inspected visually for their strength and six signs of disease, only K-wing had prevalence above 5% (40/55 colonies = 72.7%). Similar to an earlier study, screenings again confirmed the absence of V. destructor, small hive beetles Aethina tumida (Murray), tracheal mites Acarapis woodi (Rennie), and Tropilaelaps spp. ectoparasitic mites. Of a subset of 23 colonies screened molecularly for viruses, none had Israeli acute paralysis virus, Kashmir bee virus, or sacbrood virus. Sixteen of 23 colonies (70.0%) were positive for black queen cell virus, and 21 (91.3%) had some evidence for deformed wing virus. No N. ceranae was detected in molecular screens of 55 colonies, although it is possible extremely low intensity infections exist; the more familiar N. apis was found in 53 colonies (96.4%). Under these conditions, K-wing was associated (positively) with colony strength; however, viruses and N. apis were not. Furthermore, black queen cell virus was positively and negatively associated with K-wing and deformed wing virus, respectively. Newfoundland honey bee colonies are thus free of several invasive parasites that plague operations in other parts of the world, and they provide a unique research arena to study independent pathology of the parasites that are present.

Protection and Performance

We examine the linkages between import policy and export performance, extending classic macroeconomic trade effects to more recent concepts from the modern literature on gravity models. We also examine these effects empirically with a panel of global and bilateral trade spanning 15 years. Our emphasis on the role of import policy (i.e. tariffs) of exporters as an explanation of trade volumes contrasts with the recent emphasis on importer policy in the gravity literature. It also reinforces the growing body of evidence on the importance of economic environmental (policy and infrastructure) conditions in explaining relative export performance and is in line with the literature on global value chains.

Experimental infection of the pig with Mycobacterium ulcerans: a novel model for studying the pathogenesis of Buruli ulcer disease.

BACKGROUND Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available. METHODOLOGY/PRINCIPAL FINDINGS For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2 × 10(7) and 2 × 10(6) colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans. CONCLUSION/SIGNIFICANCE Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions.