Biology subjects

Wolf, E.

Publications and source records attributed to Wolf, E..

42 records · Page 3Linked to original sources

bioRxiv2020

IM30 IDPs form a membrane protective carpet upon super-complex disassembly

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated to stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins.

biophysics↗

bioRxiv2020

Chronic hyperglycaemia drives functional impairment of lymphocytes in diabetic INSC94Y transgenic pigs

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favourable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-transgenic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohaemagglutinin (PHA). Using label-free LC-MS/MS, a total of 2,704 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype in diabetics. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycaemia.

immunology↗

bioRxiv2020

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival

Xenotransplantation using pig organs has achieved survival times of more than 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNF were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

microbiology↗

bioRxiv2020

Resources for genome editing in livestock: Cas9-expressing chickens and pigs

Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons. Significance statementGenome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

molecular biology↗

bioRxiv2019

A central role of glutamine in Chlamydia infection

Obligate intracellular bacteria like Chlamydia trachomatis undergo a complex developmental cycle between infectious non-replicative (EBs) and non-infectious replicative (RBs) forms. EBs shortly after entering a host cell transform to RBs, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell it is currently unknown how the transition from EBs to RBs is initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine by the bacteria is critical to initiate EB-RB transition. These bacteria utilize glutamine to synthesize cell wall peptidoglycan which has recently been detected in the septa of replicating intracellular Chlamydia. The increased requirement for glutamine in infected cells is achieved by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine was effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limited growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 knockout mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infective strategies.

microbiology↗

bioRxiv2019

Cellular and Molecular Probing of Intact Transparent Human Organs

Optical tissue transparency permits cellular and molecular investigation of complex tissues in 3D, a fundamental need in biomedical sciences. Adult human organs are particularly challenging for this approach, owing to the accumulation of dense and sturdy molecules in decades-aged human tissues. Here, we introduce SHANEL method utilizing a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to generate the first intact transparent adult human brain and kidney, and perform 3D histology using antibodies and dyes in centimeters depth. Thereby, we revealed structural details of sclera, iris and suspensory ligament in the human eye, and the vessels and glomeruli in the human kidney. We also applied SHANEL on transgenic pig organs to map complex structures of EGFP expressing beta cells in >10 cm size pancreas. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of intact large mammalian organs.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC=\"FIGDIR/small/643908v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (89K):\norg.highwire.dtl.DTLVardef@14fea50org.highwire.dtl.DTLVardef@1a04274org.highwire.dtl.DTLVardef@6ae1fforg.highwire.dtl.DTLVardef@11b31b0_HPS_FORMAT_FIGEXP M_FIG C_FIG Supplementary Movies of SHANEL are available at http://discotechnologies.org/SHANEL/

cell biology↗