Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Detergents enable the purification of membrane proteins and are indispensable reagents instructural biology. Even though a large variety of detergents have been developed in the lastcentury, the challenge remains to identify guidelines that allowfine-tuning of detergents forindividual applications in membrane protein research. Addressing this challenge, here weintroduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS)reveals that the modular OGD architecture offers the ability to control protein purificationand to preserve interactions with native membrane lipids during purification. In addition to abroad range of bacterial membrane proteins, OGDs also enable the purification and analysisof a functional G-protein coupled receptor (GPCR). Moreover, given the modular design ofthese detergents, we anticipatefine-tuning of their properties for specific applications instructural biology. Seen from a broader perspective, this represents a significant advance forthe investigation of membrane proteins and their interactions with lipids

Issues Around Researching OHS of Samoan Migrant Workers

Workers from Pacific nations constitute a substantial proportion of the labour force in NZ, particularly in Auckland, which has one of the largest concentrations of Pacific Island workers in the world. Samoans constitute the largest Pacific ethnic group in NZ, comprising 131,103 or 49% of the resident Pacific population (265,974) (Statistics NZ, 2010). However, Pacific Island workers in NZ are typically employed in low paid, precarious, hazardous work that often has little chance of advancement. There is also some evidence that Pacific Island workers are over­represented in NZ’s work­related injury and illness statistics (Allen & Clarke, 2006). While occupational health and safety (OHS) of Pacific Island migrant workers highlights a number of issues, studies often provide inadequate explanations of what exactly is occurring or fully capture the working experiences of Pacific Island migrant workers. This paper reports on the initial work undertaken as part of an international collaborative study located in Samoa and NZ, aimed at investigating the OHS experiences of Samoan migrant workers. In particular, the paper presents a multi­layered framework and a set of research principles that can be used to illuminate often inaccessible populations located in changing working and living environments. Finally, this study exemplifies the complex issues surrounding the migrant workers’ health and safety, workers’ compensation and rehabilitation

Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803

Members of the phylum Bacteroidetes are abundant in many marine ecosystems and are known to have a pivotal role in the mineralization of complex organic substrates such as polysaccharides and proteins. We studied the decomposition of the algal glycans laminarin and alginate by 'Gramella forsetii' KT0803, a bacteroidetal isolate from North Sea surface waters. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed two large polysaccharide utilization loci (PULs) that were specifically induced, one by alginate and the other by laminarin. These regulons comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, carbohydrate-active enzymes and hypothetical proteins. Besides, several glycan-specific TonB-dependent receptors and SusD-like substrate-binding proteins were expressed also in the absence of polysaccharide substrates, suggesting an anticipatory sensing function. Genes for the utilization of the beta-1,3-glucan laminarin were found to be co-regulated with genes for glucose and alpha-1,4-glucan utilization, which was not the case for the non-glucan alginate. Strong syntenies of the PULs of 'G. forsetii' with similar loci in other Bacteroidetes indicate that the specific response mechanisms of 'G. forsetii' to changes in polysaccharide availability likely apply to other Bacteroidetes. Our results can thus contribute to an improved understanding of the ecological niches of marine Bacteroidetes and their roles in the polysaccharide decomposition part of carbon cycling in marine ecosystems

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Delta-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoARha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharide

Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions.

Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host-microbe associations. However, how host-symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host-microbe interactions can be quite variable, even between closely related systems

A long-term follow-up of safety and clinical efficacy of NTCELL® [Immunoprotected (Alginate-encapsulated) porcine choroid plexus cells for xenotransplantation] in patients with Parkinson's disease.

INTRODUCTION: In 2019, we published the results of a Phase IIb randomized controlled trial of putaminal encapsulated porcine choroid plexus cell (termed NTCELL®) administration in patients with Parkinson's disease. This study failed to meet its primary efficacy end-point of a change in UPDRS part III score in the 'off' state at 26-weeks post-implant. However, a number of secondary end-points reached statistical significance. We questioned whether with longer follow-up, clinically significant improvements would be observed. For this reason, we decided to follow-up all patients periodically to week 104. Herein, we report the results of this long-term follow-up. METHODS: All 18 patients included in the original study were periodically re-assessed at weeks 52, 78 and 104 post-implant. At each time-point, motor and non-motor function, quality of life and levodopa equivalent daily dose was assessed using a standardized testing battery. RESULTS: At week 104, no significant differences in UPDRS part III scores in the 'off' state were observed in any of the treatment groups compared to baseline. Only a single serious adverse event - hospitalisation due to Parkinson's disease rigidity not responding to changes in medications - was considered potentially related to the implant procedure. There was no evidence of xenogeneic viral transmission. CONCLUSION: Un-blinded, long-duration follow-up to week 104 post-implantation showed no evidence that putaminal NTCELL® administration produces significant clinical benefit in patients with moderately advanced Parkinson's disease

Issues Around Researching OHS of Samoan Migrant Workers

Workers from Pacific nations constitute a substantial proportion of the labour force in NZ, particularly in Auckland, which has one of the largest concentrations of Pacific Island workers in the world. Samoans constitute the largest Pacific ethnic group in NZ, comprising 131,103 or 49% of the resident Pacific population (265,974) (Statistics NZ, 2010). However, Pacific Island workers in NZ are typically employed in low paid, precarious, hazardous work that often has little chance of advancement. There is also some evidence that Pacific Island workers are over­represented in NZ’s work­related injury and illness statistics (Allen & Clarke, 2006). While occupational health and safety (OHS) of Pacific Island migrant workers highlights a number of issues, studies often provide inadequate explanations of what exactly is occurring or fully capture the working experiences of Pacific Island migrant workers. This paper reports on the initial work undertaken as part of an international collaborative study located in Samoa and NZ, aimed at investigating the OHS experiences of Samoan migrant workers. In particular, the paper presents a multi­layered framework and a set of research principles that can be used to illuminate often inaccessible populations located in changing working and living environments. Finally, this study exemplifies the complex issues surrounding the migrant workers’ health and safety, workers’ compensation and rehabilitation

Approaching the uncultured endosymbiont of Riftia pachyptila by physiological proteomics

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of AAAS for personal use, not for redistribution. The definitive version was published in Science 315 (2007): 247-250, doi:10.1126/science.1132913.The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute ~12% of the total cytosolic proteome, highlighting the essential role of these enzymes for the symbiont’s energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid (TCA) cycle in addition to the previously identified Calvin cycle for CO2 fixation.This work was supported by the DFG, grant Schw595/3-1. Other funding sources were: NSF (OCE 04-52333) and NASA Astrobiology Institute (NNA04CC04A) for SMS, MH: postdoctoral scholarship from WHOI, HF: Academic Senate (RF811S and RE518S)

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hinzke, T., Kleiner, M., Meister, M., Schlueter, R., Hentschker, C., Pane-Farre, J., Hildebrandt, P., Felbeck, H., Sievert, S. M., Bonn, F., Voelker, U., Becher, D., Schweder, T., & Markert, S. Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis. Elife, 10, (2021): e58371, https://doi.org/10.7554/eLife.58371.The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.This work was supported by the German Research Foundation DFG (grant MA 6346/2–1 to SM), fellowships of the Institute of Marine Biotechnology Greifswald (TH, MM), a German Academic Exchange Service (DAAD) grant (TH), the NC State Chancellor’s Faculty Excellence Program Cluster on Microbiomes and Complex Microbial Communities (MK), the USDA National Institute of Food and Agriculture, Hatch project 1014212 (MK), the U.S. National Science Foundation (grants OCE-1131095 and OCE-1559198 to SMS), and The WHOI Investment in Science Fund (to SMS). We furthermore acknowledge support for article processing charges from the DFG (Grant 393148499) and the Open Access Publication Fund of the University of Greifswald