Cell wall composition and biomass saccharification potential of Sida hermaphrodita differ between genetically distant accessions.

Due to its ample production of lignocellulosic biomass, Sida hermaphrodita (Sida), a perennial forb, is considered a valuable raw material for biorefinery processes. The recalcitrant nature of Sida lignocellulosic biomass towards pretreatment and fractionation processes has previously been studied. However, Sida is a non-domesticated species and here we aimed at expanding the potential of such plants in terms of their processability for downstream processes by making use of the natural variety of Sida. To achieve this goal, we established a collection comprising 16 different Sida accessions obtained from North America and Europe. First, we asked whether their cell wall characteristics are reflected in genetic distance or geographical distribution, respectively. A genotyping-by-sequencing (GBS) analysis resulting in a phylogenic tree based on 751 Single Nucleotide Polymorphisms (SNPs), revealed a high genetic diversity and a clear separation between accessions collected in North America and Europe. Further, all three North American accessions were separated from each other. Of the eleven European accessions, five form individual groups and six others belong to a single group. Clonal plants of seven selected accessions of American and European origin were produced and cultivated under greenhouse conditions and the resulting plant material was used for in-depth wet-chemical and spectroscopic cell wall characterization. Two accessions with contrasting cell wall characteristics were then selected and processed using the OrganoCat technology. Results of the different product yields and chemical compositions are reported. Overall, cell wall analyses revealed contrasting clusters regarding these main components between the accessions that can be related to genetic and, partly, geographical distance. Phenotypically, the accessions clustered into two groups that are not entirely overlapping with geographical origin. These results can be the basis for a targeted selection or cultivation of Sida accessions for biorefinery approaches.

DARPins bind their cytosolic targets after having been translocated through the protective antigen pore of anthrax toxin.

Intracellular protein-protein interactions in aberrant signaling pathways have emerged as a prime target in several diseases, particularly cancer. Since many protein-protein interactions are mediated by rather flat surfaces, they can typically not be interrupted by small molecules as they require cavities for binding. Therefore, protein drugs might be developed to compete with undesired interactions. However, proteins in general are not able to translocate from the extracellular side to the cytosolic target site by themselves, and thus an efficient protein translocation system, ideally combining efficient translocation with receptor specificity, is in high demand. Anthrax toxin, the tripartite holotoxin of Bacillus anthracis, is one of the best studied bacterial protein toxins and has proven to be a suitable candidate for cell-specific translocation of cargoes in vitro and in vivo. Our group recently developed a retargeted protective antigen (PA) variant fused to different Designed Ankyrin Repeat Proteins (DARPins) to achieve receptor specificity, and we incorporated a receptor domain to stabilize the prepore and prevent cell lysis. This strategy had been shown to deliver high amounts of cargo DARPins fused behind the N-terminal 254 amino acids of Lethal Factor (LFN). Here, we established a cytosolic binding assay, demonstrating the ability of DARPins to refold in the cytosol and bind their target after been translocated by PA.

Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation.

OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

Synthesis and Concomitant Assembly of Adeno-Associated Virus-like Particles in Escherichia coli.

Virus-like particles (VLPs) have been used for numerous pharmaceutical applications, particularly vaccination and drug delivery. Recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy, has been proposed as a vaccine scaffold, but high production costs limit its use. Here we establish intracellular production of AAV VLPs in Escherichia coli. VP3 capsid proteins of AAV serotype 5 (AAV5) were expressed, and VLPs were readily purified. The correct assembly was confirmed by ELISA with an intact-capsid AAV5 antibody and an AAVR domain as well as by atomic force microscopy. Biological functionality was demonstrated with a HeLa cell internalization assay. Coexpression of the assembly-activating protein (AAP) of AAV5 in E. coli improved capsid yield. This work provides the first evidence that AAV VLPs form in E. coli, opening new opportunities for production and exploration of AAV VLPs for biomedical applications.

Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin.

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Meltwater sediment transport as the dominating process in mid-latitude trough mouth fan formation.

Trough mouth fans comprise the largest sediment deposits along glaciated margins, and record Pleistocene climate changes on a multi-decadal time scale. Here we present a model for the formation of the North Sea Fan derived from detailed horizon and attribute interpretations of high-resolution processed 3D seismic reflection data. The interpretation shows that stacked channel-levee systems form up to 400 m thick sedimentary sequences. The channels are elongated and can be traced from the shelf edge towards the deep basin for distances of >150 km, and document long-distance sediment transport in completely disintegrated water-rich turbidite flows. Downslope sediment transport was a continuous process during shelf-edge glaciations, reaching accumulation rates of 100 m/kyr. Our data highlight that exceptionally large volumes of meltwater may discharge to the slopes of trough mouth fans and trigger erosive turbidite flows. We conclude that freshwater supply is likely an underestimated factor for sedimentary processes during glacial cycles.

Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery.

BACKGROUND: To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. RESULTS: Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. CONCLUSION: We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol.

Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4ß7 and Development of Colitis in Mice.

BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.

Comparison of two measurement strategies to obtain the residence time distribution in combustion chambers using tunable diode laser absorption spectroscopy.

The residence time distribution (RTD) of fluid elements in combustion chambers is a key feature of the flow field, and its knowledge is therefore important for the design and improvement of combustion systems. The hostile yet sensitive environment of burners, in particular with high particle loads of solid-fuel firing, impede direct access to this quantity. Two strategies to obtain the RTD based on hydrogen chloride injection with subsequent detection by tunable diode laser absorption spectroscopy are directly compared and applied to gas and solid-fuel combustion under two different combustion modes. Through the direct comparison of this work, the experimentally more challenging and thus rarely utilized pulse injection was found to be superior to the commonly used step injection.

Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization.

Biomolecules have many properties that make them promising for intracellular therapeutic applications, but delivery remains a key challenge because large biomolecules cannot easily enter the cytosol. Furthermore, quantification of total intracellular versus cytosolic concentrations remains demanding, and the determination of delivery efficiency is thus not straightforward. In this review, we discuss strategies for delivering biomolecules into the cytosol and briefly summarize the mechanisms of uptake for these systems. We then describe commonly used methods to measure total cellular uptake and, more selectively, cytosolic localization, and discuss the major advantages and drawbacks of each method. We critically evaluate methods of measuring "cell penetration" that do not adequately distinguish total cellular uptake and cytosolic localization, which often lead to inaccurate interpretations of a molecule's cytosolic localization. Finally, we summarize the properties and components of each method, including the main caveats of each, to allow for informed decisions about method selection for specific applications. When applied correctly and interpreted carefully, methods for quantifying cytosolic localization offer valuable insight into the bioactivity of biomolecules and potentially the prospects for their eventual development into therapeutics.

BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous carcinoma.

Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development.

Author Correction: Glacigenic sedimentation pulses triggered post-glacial gas hydrate dissociation.

The original version of this Article contained an error in the second sentence of the Abstract, which incorrectly read 'They are stable under high pressure and low, but react sensitively to environmental changes.' The correct version adds 'temperature' after 'low'. This has been corrected in both the PDF and HTML versions of the Article.

Glacigenic sedimentation pulses triggered post-glacial gas hydrate dissociation.

Large amounts of methane are stored in continental margins as gas hydrates. They are stable under high pressure and low, but react sensitively to environmental changes. Bottom water temperature and sea level changes were considered as main contributors to gas hydrate dynamics after the last glaciation. However, here we show with numerical simulations that pulses of increased sedimentation dominantly controlled hydrate stability during the end of the last glaciation offshore mid-Norway. Sedimentation pulses triggered widespread gas hydrate dissociation and explains the formation of ubiquitous blowout pipes in water depths of 600 to 800 m. Maximum gas hydrate dissociation correlates spatially and temporally with the formation or reactivation of pockmarks, which is constrained by radiocarbon dating of Isorropodon nyeggaensis bivalve shells. Our results highlight that rapid changes of sedimentation can have a strong impact on gas hydrate systems affecting fluid flow and gas seepage activity, slope stability and the carbon cycle.