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1.
  • Apelgren, Peter, et al. (författare)
  • Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.
  • 2017
  • Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 12:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.
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3.
  • Gillman, Anna, et al. (författare)
  • Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards
  • 2015
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 81:7, s. 2378-2383
  • Tidskriftsartikel (refereegranskat)abstract
    • Influenza A virus (IAV) has its natural reservoir in wild waterfowl and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate (OC)), stockpiled as Tamiflu® for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may there exert evolutionary pressure on avian IAV in waterfowl, resulting in development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo Mallard (Anas platyrhynchos) study we tested if an OC-resistant avian IAV strain (A(H1N1)/NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected Mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission in 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV, induced by OC exposure of the natural host, can persist in absence of the drug. Thus, there is a risk that human pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir resistant pandemic IAV would be a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment and resistance surveillance of IAV in wild birds.
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4.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
  • 2019
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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5.
  • Wang, Guokun, 1988, et al. (författare)
  • RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
  • 2019
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:19, s. 9324-9332
  • Tidskriftsartikel (refereegranskat)abstract
    • The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.
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6.
  • Apelgren, Peter, et al. (författare)
  • In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink
  • 2019
  • Ingår i: Acs Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 5:5, s. 2482-2490
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 X 5 X 1 mm(3)) containing human nasal chondrocytes (10 M mL(-1)) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 +/- 13.8 cells per mm(2) observed after 30 days and 85.6 +/- 30.0 cells per mm(2) at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair.
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7.
  • Dietrich, Franciele, et al. (författare)
  • Effect of storage and preconditioning of healing rat Achilles tendon on structural and mechanical properties
  • 2021
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Tendon tissue storage and preconditioning are often used in biomechanical experiments and whether this generates alterations in tissue properties is essential to know. The effect of storage and preconditioning on dense connective tissues, like tendons, is fairly understood. However, healing tendons are unlike and contain a loose connective tissue. Therefore, we investigated if storage of healing tendons in the fridge or freezer changed the mechanical properties compared to fresh tendons, using a pull-to-failure or a creep test. Tissue morphology and cell viability were also evaluated. Additionally, two preconditioning levels were tested. Rats underwent Achilles tendon transection and were euthanized 12 days postoperatively. Statistical analyzes were done with one-way ANOVA or Student’s t-test. Tissue force and stress were unaltered by storage and preconditioning compared to fresh samples, while high preconditioning increased the stiffness and modulus (p ≤ 0.007). Furthermore, both storage conditions did not modify the viscoelastic properties of the healing tendon, but altered transverse area, gap length, and water content. Cell viability was reduced after freezing. In conclusion, preconditioning on healing tissues can introduce mechanical data bias when having extensive tissue strength diversity. Storage can be used before biomechanical testing if structural properties are measured on the day of testing.
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8.
  • Kanagarajan, Selvaraju, et al. (författare)
  • Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
  • 2021
  • Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
  • Tidskriftsartikel (refereegranskat)abstract
    • Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
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9.
  • Säljö, Karin, 1981, et al. (författare)
  • Successful engraftment, vascularization, and In vivo survival of 3D-bioprinted human lipoaspirate-derived adipose tissue
  • 2020
  • Ingår i: Bioprinting. - : Elsevier BV. - 2405-8866. ; 17
  • Tidskriftsartikel (refereegranskat)abstract
    • Autologous fat grafting is commonly used for correction of soft-tissue deformities, despite a high rate of graft resorption and nutrition-supply challenges. Three-dimensional (3D)-bioprinting techniques enable tailor-made architecture of grafts and promote vascularization. In recent years, the importance of adipose tissue-derived stromal/stem cells (ASCs) for graft survival has become evident. This study investigated the printability of mechanically processed lipoaspirate containing ASCs, as well as in vivo survival and neovascularisation of the 3D-bioprinted grafts. Human lipoaspirate-derived adipose tissue was 3D bioprinted in alginate/nanocellulose bioink, implanted into nude mice, and harvested at days 3, 7, and 30, respectively. The processed lipoaspirate showed high viability and good printability when combined with alginate/nanocellulose, and the 3D-bioprinted grafts contained intact vascular structures and a high density of mature adipocytes before and after engraftment. After 30 days in vivo, novel blood vessels were present on the graft surface, showing signs of angiogenesis into the graft, as well as vascularization in the centre of the tissue. Moreover, histologic and immunohistochemical characterisation confirmed the presence of potential ASCs during the first week in vivo. These results demonstrated that human lipoaspirate-derived adipose tissue showed high printability, survived 3D bioprinting and engraftment in vivo, and displayed macroscopic and microscopic evidence of vascularization.
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10.
  • Gullfot, Fredrika, 1967- (författare)
  • Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
  • 2009
  • Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.
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11.
  • Huang, Mingtao, 1984, et al. (författare)
  • Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production
  • 2018
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:47, s. E11025-E11032
  • Tidskriftsartikel (refereegranskat)abstract
    • Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
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12.
  • Cardemil, Carina, et al. (författare)
  • Strontium-doped calcium phosphate and hydroxyapatite granules promote different inflammatory and bone remodelling responses in normal and ovariectomised rats.
  • 2013
  • Ingår i: PLosOne. - : Public Library of Science (PLoS). - 1932-6203. ; 8:12
  • Tidskriftsartikel (refereegranskat)abstract
    • The healing of bone defects may be hindered by systemic conditions such as osteoporosis. Calcium phosphates, with or without ion substitutions, may provide advantages for bone augmentation. However, the mechanism of bone formation with these materials is unclear. The aim of this study was to evaluate the healing process in bone defects implanted with hydroxyapatite (HA) or strontium-doped calcium phosphate (SCP) granules, in non-ovariectomised (non-OVX) and ovariectomised (OVX) rats. After 0 (baseline), six and 28d, bone samples were harvested for gene expression analysis, histology and histomorphometry. Tumour necrosis factor-α (TNF-α), at six days, was higher in the HA, in non-OVX and OVX, whereas interleukin-6 (IL-6), at six and 28d, was higher in SCP, but only in non-OVX. Both materials produced a similar expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). Higher expression of osteoclastic markers, calcitonin receptor (CR) and cathepsin K (CatK), were detected in the HA group, irrespective of non-OVX or OVX. The overall bone formation was comparable between HA and SCP, but with topological differences. The bone area was higher in the defect centre of the HA group, mainly in the OVX, and in the defect periphery of the SCP group, in both non-OVX and OVX. It is concluded that HA and SCP granules result in comparable bone formation in trabecular bone defects. As judged by gene expression and histological analyses, the two materials induced different inflammatory and bone remodelling responses. The modulatory effects are associated with differences in the spatial distribution of the newly formed bone.
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13.
  • Karlsson, Johan, 1984, et al. (författare)
  • Stem cell homing using local delivery of plerixafor and stromal derived growth factor-1alpha for improved bone regeneration around Ti-implants
  • 2016
  • Ingår i: Journal of Biomedical Materials Research - Part A. - : Wiley. - 1552-4965 .- 1549-3296. ; 104:10, s. 2466-2475
  • Tidskriftsartikel (refereegranskat)abstract
    • Triggering of the early healing events, including the recruitment of progenitor cells, has been suggested to promote bone regeneration. In implantology, local drug release technologies could provide an attractive approach to promote tissue regeneration. In this study, we targeted the chemotactic SDF-1a/CXCR4 axis that is responsible e.g. for the homing of stem cells to trauma sites. This was achieved by local delivery of plerixafor, an antagonist to CXCR4, and/or SDF-1a from titanium implants coated with mesoporous titania thin films with a pore size of 7.5 nm. In vitro drug delivery experiments demonstrated that the mesoporous coating provided a high drug loading capacity and controlled release. The subsequent in vivo study in rat tibia showed beneficial effects with respect to bone-implant anchorage and bone-formation along the surface of the implants when plerixafor and SDF-1a were delivered locally. The effect was most prominent by the finding that the combination of the drugs significantly improved the mechanical bone anchorage. These observations suggest that titanium implants with local delivery of drugs for enhanced local recruitment of progenitor cells have the ability to promote osseointegration. This approach may provide a potential strategy for the development of novel implant treatments.
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14.
  • Cámara, Elena, 1985, et al. (författare)
  • Data mining of Saccharomyces cerevisiae mutants engineered for increased tolerance towards inhibitors in lignocellulosic hydrolysates
  • 2022
  • Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 57
  • Forskningsöversikt (refereegranskat)abstract
    • The use of renewable plant biomass, lignocellulose, to produce biofuels and biochemicals using microbial cell factories plays a fundamental role in the future bioeconomy. The development of cell factories capable of efficiently fermenting complex biomass streams will improve the cost-effectiveness of microbial conversion processes. At present, inhibitory compounds found in hydrolysates of lignocellulosic biomass substantially influence the performance of a cell factory and the economic feasibility of lignocellulosic biofuels and chemicals. Here, we present and statistically analyze data on Saccharomyces cerevisiae mutants engineered for altered tolerance towards the most common inhibitors found in lignocellulosic hydrolysates: acetic acid, formic acid, furans, and phenolic compounds. We collected data from 7971 experiments including single overexpression or deletion of 3955 unique genes. The mutants included in the analysis had been shown to display increased or decreased tolerance to individual inhibitors or combinations of inhibitors found in lignocellulosic hydrolysates. Moreover, the data included mutants grown on synthetic hydrolysates, in which inhibitors were added at concentrations that mimicked those of lignocellulosic hydrolysates. Genetic engineering aimed at improving inhibitor or hydrolysate tolerance was shown to alter the specific growth rate or length of the lag phase, cell viability, and vitality, block fermentation, and decrease product yield. Different aspects of strain engineering aimed at improving hydrolysate tolerance, such as choice of strain and experimental set-up are discussed and put in relation to their biological relevance. While successful genetic engineering is often strain and condition dependent, we highlight the conserved role of regulators, transporters, and detoxifying enzymes in inhibitor tolerance. The compiled meta-analysis can guide future engineering attempts and aid the development of more efficient cell factories for the conversion of lignocellulosic biomass.
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15.
  • Rugbjerg, Peter, 1988, et al. (författare)
  • The future of self-selecting and stable fermentations
  • 2020
  • Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 47:11, s. 993-1004
  • Forskningsöversikt (refereegranskat)abstract
    • Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production.
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16.
  • Apelgren, Peter, et al. (författare)
  • Vascularization of tissue engineered cartilage-Sequential in vivo MRI display functional blood circulation
  • 2021
  • Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 276
  • Tidskriftsartikel (refereegranskat)abstract
    • Establishing functional circulation in bioengineered tissue after implantation is vital for the delivery of oxygen and nutrients to the cells. Native cartilage is avascular and thrives on diffusion, which in turn depends on proximity to circulation. Here, we investigate whether a gridded three-dimensional (3D) bioprinted construct would allow ingrowth of blood vessels and thus prove a functional concept for vascularization of bioengineered tissue. Twenty 10 x 10 x 3-mm 3Dbioprinted nanocellulose constructs containing human nasal chondrocytes or cell-free controls were subcutaneously implanted in 20 nude mice. Over the next 3 months, the mice were sequentially imaged with a 7 T small-animal MRI system, and the diffusion and perfusion parameters were analyzed. The chondrocytes survived and proliferated, and the shape of the constructs was well preserved. The diffusion coefficient was high and well preserved over time. The perfusion and diffusion patterns shown by MRI suggested that blood vessels develop over time in the 3D bioprinted constructs; the vessels were confirmed by histology and immunohistochemistry. We conclude that 3D bioprinted tissue with a gridded structure allows ingrowth of blood vessels and has the potential to be vascularized from the host. This is an essential step to take bioengineered tissue from the bench to clinical practice.
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17.
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18.
  • Ferreira, Raphael, 1990, et al. (författare)
  • Tackling Cancer with Yeast-Based Technologies
  • 2019
  • Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 37:6, s. 592-603
  • Forskningsöversikt (refereegranskat)abstract
    • The ability to precisely engineer yeast, coupled with its genetic and metabolic similarity to tumor cells, has enabled researchers to use this organism in cancer research. Here we review advances that leveraged yeast as a model organism for studying cancer biology, including the investigation of tumorigenic mechanisms, development of advanced technologies for drug discovery, production of anticancer drugs on an industrial scale, and delivering the next generation of immunotherapies.
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19.
  • Garousi, Javad, et al. (författare)
  • Radionuclide therapy using ABD-fused ADAPT scaffold protein : Proof of Principle
  • 2021
  • Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 266
  • Tidskriftsartikel (refereegranskat)abstract
    • Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.
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20.
  • Gillman, Anna, et al. (författare)
  • Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards
  • 2015
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 81:7, s. 2378-2383
  • Tidskriftsartikel (refereegranskat)abstract
    • Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.
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21.
  • Nielsen, Jens B, 1962 (författare)
  • METABOLISM: A stress-coping strategy for yeast cells
  • 2019
  • Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 572:7768, s. 184-185
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • Stressed yeast cells take up the amino acid lysine and reprogram their metabolism to free up supplies of a stress-relieving molecule. Lysine uptake therefore increases the tolerance of yeast cells to stress. See LETTER P.249
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22.
  • Oroujeni, Maryam, PhD, 1982-, et al. (författare)
  • Preclinical Evaluation of [Ga-68]Ga-DFO-ZEGFR:2377 : A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors
  • 2018
  • Ingår i: Cells. - : MDPI. - 2073-4409. ; 7:9
  • Tidskriftsartikel (refereegranskat)abstract
    • Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide Ga-68. Stability, specificity of binding to EGFR-expressing cells, and processing of [Ga-68]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [Ga-68]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [Zr-89]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 +/- 3%) and stably labeled with Ga-68. Binding of [Ga-68]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [Ga-68]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [Zr-89]Zr-DFO-ZEGFR:2377, [Ga-68]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [Ga-68]Ga-DFO-ZEGFR:2377. In conclusion, [Ga-68]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.
  •  
23.
  • Schneider, Kara, et al. (författare)
  • Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates
  • 2022
  • Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 298:11
  • Tidskriftsartikel (refereegranskat)abstract
    • The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing.
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24.
  • Skiöld, Sara, et al. (författare)
  • Radiation-induced stress response in peripheral blood of breast cancer patients differs between patients with severe acute skin reactions and patients with no side effects to radiotherapy
  • 2013
  • Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 756:1-2, s. 152-157
  • Tidskriftsartikel (refereegranskat)abstract
    • The aim of the study was to compare the radiation-induced oxidative stress response in blood samples from breast cancer patients that developed severe acute skin reactions during the radiotherapy, with the response in blood samples from patients with no side effects. Peripheral blood was collected from 12 breast cancer patients showing no early skin reactions after radiotherapy (RTOG grade 0) and from 14 breast cancer patients who developed acute severe skin reactions (RTOG grade 3-4). Whole blood was irradiated with 0, 5 and 2000 mGy gamma-radiation and serum was isolated. The biomarker for oxidative stress, 8-oxo-dG, was analyzed in the serum by a modified ELISA. While a significant radiation-induced increase of serum 8-oxo-dG levels was observed in serum of the RTOG 0 patients, no increase was seen in serum of the RTOG 3-4 patients. The radiation induced increase in serum 8-oxo-dG levels after 5 mGy did not differ significantly from the increase observed for 2000 mGy in the RTOG 3-4 cohort, thus no dose response relation was observed. A receiver operating characteristic (ROC) value of 0.97 was obtained from the radiation-induced increase in 8-oxo-dG indicating that the assay could be used to identify patients with severe acute adverse reactions to radiotherapy. The results show that samples of whole blood from patients, classified as highly radiosensitive (RTOG 3-4) based on their skin reactions to radiotherapy, differ significantly in their oxidative stress response to ionizing radiation compared to samples of whole blood from patients with no skin reactions (RTOG 0). Extracellular 8-oxo-dG is primarily a biomarker of nucleotide damage and the results indicate that the patients with severe acute skin reactions differ in their cellular response to ionizing radiation at the level of induction of oxidative stress or at the level of repair or both.
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25.
  • Skrekas, Christos, 1990, et al. (författare)
  • Fluorescence-Activated Cell Sorting as a Tool for Recombinant Strain Screening
  • 2022
  • Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2513, s. 39-57
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • Metabolic engineering of microbial cells is the discipline of optimizing microbial metabolism to enable and improve the production of target molecules ranging from biofuels and chemical building blocks to high-value pharmaceuticals. The advances in genetic engineering have eased the construction of highly engineered microbial strains and the generation of genetic libraries. Intracellular metabolite-responsive biosensors facilitate high-throughput screening of these libraries by connecting the levels of a metabolite of interest to a fluorescence output. Fluorescent-activated cell sorting (FACS) enables the isolation of highly fluorescent single cells and thus genotypes that produce higher levels of the metabolite of interest. Here, we describe a high-throughput screening method for recombinant yeast strain screening based on intracellular biosensors and FACS.
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26.
  • Svärd, Anna, 1985-, et al. (författare)
  • Elastin levels are higher in healing tendons than in intact tendons and influence tissue compliance
  • 2020
  • Ingår i: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 34:10, s. 13409-13418
  • Tidskriftsartikel (refereegranskat)abstract
    • Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28 days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14 days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20x 10 N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28 days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. Plain Language Summary Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. When a tendon is injured and ruptured the tissue heals through scar formation. This scar tissue is different from a normal intact tendon and it is important to understand how the tendons heal. Little is known about the presence and function of elastin during healing of tendon injuries. We have shown, in animal experiments, that healing tendons have higher amounts of elastin compared to intact tendons. The elastin is also spread throughout the tissue. When we reduced the levels of this protein, we discovered altered mechanical properties of the tendon. The healing tendon can normally extend quite a lot, but after elastin removal this extensibility was less obvious. The ability of the healing tissue to extend is probably important to protect the tendon from re-injuries during the first months after rupture. We therefore propose that the tendons heal with a large amount of elastin to prevent re-ruptures during early locomotion.
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27.
  • Turanli, Beste, et al. (författare)
  • Multi-Omic Data Interpretation to Repurpose Subtype Specific Drug Candidates for Breast Cancer
  • 2019
  • Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 10:MAY
  • Tidskriftsartikel (refereegranskat)abstract
    • Triple-negative breast cancer (TNBC), which is largely synonymous with the basal-like molecular subtype, is the 5th leading cause of cancer deaths for women in the United States. The overall prognosis for TNBC patients remains poor given that few treatment options exist; including targeted therapies (not FDA approved), and multi-agent chemotherapy as standard-of-care treatment. TNBC like other complex diseases is governed by the perturbations of the complex interaction networks thereby elucidating the underlying molecular mechanisms of this disease in the context of network principles, which have the potential to identify targets for drug development. Here, we present an integrated "omics" approach based on the use of transcriptome and interactome data to identify dynamic/active protein-protein interaction networks (PPINs) in TNBC patients. We have identified three highly connected modules, EED, DHX9, and AURKA, which are extremely activated in TNBC tumors compared to both normal tissues and other breast cancer subtypes. Based on the functional analyses, we propose that these modules are potential drivers of proliferation and, as such, should be considered candidate molecular targets for drug development or drug repositioning in TNBC. Consistent with this argument, we repurposed steroids, anti-inflammatory agents, anti-infective agents, cardiovascular agents for patients with basal-like breast cancer. Finally, we have performed essential metabolite analysis on personalized genome-scale metabolic models and found that metabolites such as sphingosine-1-phosphate and cholesterol-sulfate have utmost importance in TNBC tumor growth.
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28.
  • Zirk, Katrin, et al. (författare)
  • Vectorization of splice-correcting oligonucleotides with cell-penetrating peptides
  • 2013
  • Ingår i: Chimica oggi. - 0392-839X .- 1973-8250. ; 31:2, s. 12-15
  • Tidskriftsartikel (refereegranskat)abstract
    • Personalized medicine approaches based on different gene therapy settings have gained much attention lately. In order to enforce successful gene therapy, genetic material needs to be delivered into cells. Nucleic acids and their analogues are unable to do so and thus require assistance to reach their site of action residing in the cytoplasm or nucleus. Here we give a short review on recent advancements in cell-penetrating peptide mediated delivery of splice-correcting oligonucleotides. We report on different cell-penetrating peptides applied for vectorization of splice-correcting oligonucleotides using both covalent conjugation and non-covalent nanoparticle formation approach. While covalent conjugation has gained extensive interest, there have also been great advances in non-covalent complex formation.
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29.
  • Iseri, Emre (författare)
  • Microfluidic Compartmentalization for Smart Materials, Medical Diagnostics and Cell Therapy
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The organisation of fluids in small compartments is ubiquitous in nature, such as in the cellular composition of all life. This work explores several engineering avenues where microscale fluid compartmentalization can bring novel material properties or novel functionality in life sciences or medicine. Here, we introduce four unique compartmentalization methods: 1) 3D fluid self-organisation in microscaffolds (FLUID3EAMS), 2) 2D microcapillary arrays on a dipstick (Digital Dipstick), 3) a sliding microfluidic platform with cross-flow (Slip-X-Chip), and 4) compartmentalization by cutting of soft solid matter (Solidify & Cut). These methods were used in a wide range of applications. Within the area of smart materials, we applied FLUID3EAMS to synthesize materials with temperature-tuneable permeability and surface energy and to establish, in a well-controlled fashion, tissue-like materials in the form of 3D droplet interface bilayer networks. Solidify & Cut was used to form soft composites with a new type of magnetic behaviour, rotation-induced ferromagnetism, that allows easy reprogramming of the magnetization of magnetopolymers. Within the area of medical diagnostics, we applied Digital Dipstick to perform rapid digital bacterial culture in a dipstick format and obtained clinically relevant diagnostic results on samples from patients with a urinary tract infection. Furthermore, Slip-X-Chip enables particle concentration and washing as new functions in sliding microfluidic platforms, which significantly expands their potential application area. Finally, within the area of cell therapy, we explored the microencapsulation of high concentrations of therapeutic cells and presented a novel technique to fabricate core-shell microcapsules by exploiting the superior material properties of spider silk membranes. 
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30.
  • Langer, Krzysztof, et al. (författare)
  • Rapid production and recovery of cell spheroids by automated droplet microfluidics
  • 2019
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.
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31.
  • Bao, Jichen, 1988 (författare)
  • Engineering the Secretory Pathway for Recombinant Protein Secretion in Saccharomyces cerevisiae
  • 2018
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • In the past few decades there has been an increasing demand of biopharmaceutical proteins in the market. Several types of cell factories are applied to produce different pharmaceutical proteins. However, manufacturers prefer to use a few favorable biological platforms to undertake the production tasks with low cost, high productivity and proper post-translational modifications. The yeast Saccharomyces cerevisiae is one of these preferred cell factories as it has many advantages. There are several reports on improvement of recombinant protein production by S. cerevisiae through rational engineering of different stages of the protein secretion pathway. In the first story of this thesis, we engineered protein anterograde trafficking by over-expression of SEC16 to increase the secretory capacity of yeast. We performed bioreactor fermentation to further characterize the engineered strains, and we analysis the reactive oxygen species accumulation, endoplasmic reticulum exit sites, the amount of endoplasmic reticulum membranes of the strains, etc. In the second story, we engineered the retrograde trafficking by over-expression of GLO3 and GCS1 to further increase the secretory capacity of yeast based on the strain constructed in the first story. Physiological changes in the engineered strains were analyzed. We also performed additional experiments to investigate the changes in the amount of endoplasmic reticulum membranes and reactive oxygen species accumulation. In the third story, we performed a systems level analysis of the high α-amylase production strains, which were screened from UV mutation in the previous study. We identified common regulation patterns and hereby we could specify some general rules for efficient protein secretion. Last, we reported an efficient yeast secretion assay platform for biomedical and biotechnological applications. This platform is responsive to secretory disturbances from both chemicals and proteins and is potentially applicable to drug screening and the selection of cell engineering targets for protein production.
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32.
  • Björkeroth, Johan, 1990, et al. (författare)
  • Proteome reallocation from amino acid biosynthesis to ribosomes enables yeast to grow faster in rich media
  • 2020
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:35, s. 21804-21812
  • Tidskriftsartikel (refereegranskat)abstract
    • Several recent studies have shown that the concept of proteome constraint, i.e., the need for the cell to balance allocation of its proteome between different cellular processes, is essential for ensuring proper cell function. However, there have been no attempts to elucidate how cells' maximum capacity to grow depends on protein availability for different cellular processes. To experimentally address this, we cultivated Saccharomyces cerevisiae in bioreactors with or without amino acid supplementation and performed quantitative proteomics to analyze global changes in proteome allocation, during both anaerobic and aerobic growth on glucose. Analysis of the proteomic data implies that proteome mass is mainly reallocated from amino acid biosynthetic processes into translation, which enables an increased growth rate during supplementation. Similar findings were obtained from both aerobic and anaerobic cultivations. Our findings show that cells can increase their growth rate through increasing its proteome allocation toward the protein translational machinery.
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33.
  • Kolber, Natalie S., et al. (författare)
  • Orthogonal translation enables heterologous ribosome engineering in E. coli
  • 2021
  • Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 12:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.
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34.
  • Mapelli, Valeria, 1978, et al. (författare)
  • Biotechnology for production of bioactive seleno compounds and study of their influence on mouse metabolome
  • 2011
  • Ingår i: Natural Products Chemistry, Biology and Medicine IV Aug 28 - Sept 2, Acquafredda di Maratea, Italy.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Organic seleno compounds are recognized as effective anti-oxidant agents and their bioactive role in prevention of certain forms of cancer has been suggested via in vitro studies and clinical trials. Among these compounds, Seleno-methyselenocysteine (SeMCys) and γ-glutamyl-SeMCys (γ-glu-SeMCys) are the most bioactive and the latter is the preferred storage form of selenium in Se-accumulator plants thanks to their Se-methyltransferase. Therefore, Se-accumulator edible plants such as Brassicaceae and Allioideae are the main source of SeMCys and γ-glu-SeMCys in the human diet. However, seasonal and environmental factors highly affect the content and the bioavailability of these bioactive compounds. A strategy to by-pass this problem and prevent selenium shortage in human diet is the production of Se-enriched yeast (Se-yeast) to be used as food supplement. In this work we show a biotechnological approach for production Se-yeast featured by higher content of SeMCys and γ-glu-SeMCys. Coupling of metabolic engineering and bioprocess optimization resulted in a Se-yeast with 24-fold increase of SeMCys levels, compared to commercial Se-yeast. The actual effect of the produced yeast has been evaluated in an animal study. In particular, as specific Se-compounds are known to activate phase II enzymes via the electrophile-responsive element (EpRE), this response was studied in transgenic mice expressing the luciferase gene under EpRE control. We observed no effect on regulation of EpRE, either overall or hepatic, by the different Se-supplements. Paradoxically, a decrease was observed in intestinal EpRE transactivation upon supplementation of the Se-yeast produced. The overall effect of the diet supplemented with Se-yeast on mouse metabolism is currently being evaluated by metabolome analysis of liver samples from the transgenic mice.
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35.
  • Mormino, Maurizio, 1988, et al. (författare)
  • Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae
  • 2021
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 21:6
  • Tidskriftsartikel (refereegranskat)abstract
    • Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-coenzyme A, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid-producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid-tolerant strain libraries.
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36.
  • Mukherjee, Vaskar, 1986, et al. (författare)
  • A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae
  • 2021
  • Ingår i: mSystems. - 2379-5077. ; 6:4
  • Tidskriftsartikel (refereegranskat)abstract
    • CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions, and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for the industrial conversion of lignocellulosic biomasses. Tolerance to acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of .9,000 strains, where .98% of all essential and respiratory growth-essential genes were targeted with multiple guide RNAs (gRNAs). The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where high-resolution CRISPRi library screening paves the way to understanding and bioengineering the robustness of yeast against acetic acid stress.
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37.
  • Raghavendran, Vijayendran, 1976, et al. (författare)
  • The protective role of intracellular glutathione in Saccharomyces cerevisiae during lignocellulosic ethanol production
  • 2020
  • Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 10:1
  • Tidskriftsartikel (refereegranskat)abstract
    • To enhance the competitiveness of industrial lignocellulose ethanol production, robust enzymes and cell factories are vital. Lignocellulose derived streams contain a cocktail of inhibitors that drain the cell of its redox power and ATP, leading to a decrease in overall ethanol productivity. Many studies have attempted to address this issue, and we have shown that increasing the glutathione (GSH) content in yeasts confers tolerance towards lignocellulose inhibitors, subsequently increasing the ethanol titres. However, GSH levels in yeast are limited by feedback inhibition of GSH biosynthesis. Multidomain and dual functional enzymes exist in several bacterial genera and they catalyse the GSH biosynthesis in a single step without the feedback inhibition. To test if even higher intracellular glutathione levels could be achieved and if this might lead to increased tolerance, we overexpressed the genes from two bacterial genera and assessed the recombinants in simultaneous saccharification and fermentation (SSF) with steam pretreated spruce hydrolysate containing 10% solids. Although overexpressing the heterologous genes led to a sixfold increase in maximum glutathione content (18 µmol gdrycellmass−1) compared to the control strain, this only led to a threefold increase in final ethanol titres (8.5 g L− 1). As our work does not conclusively indicate the cause-effect of increased GSH levels towards ethanol titres, we cautiously conclude that there is a limit to cellular fitness that could be accomplished via increased levels of glutathione.
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38.
  • Rems, Lea, et al. (författare)
  • Cell electrofusion using nanosecond electric pulses
  • 2013
  • Ingår i: Scientific Reports. - : Macmillan Publishers Ltd.. - 2045-2322. ; 3
  • Tidskriftsartikel (refereegranskat)abstract
    • Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
  •  
39.
  • Sepehri, Sobhan, 1986, et al. (författare)
  • Volume-amplified magnetic bioassay integrated with microfluidic sample handling and high-Tc SQUID magnetic readout
  • 2018
  • Ingår i: APL Bioengineering. - : AIP Publishing. - 2473-2877. ; 2:1
  • Tidskriftsartikel (refereegranskat)abstract
    • A bioassay based on a high-Tc superconducting quantum interference device (SQUID) reading out functionalized magnetic nanoparticles (fMNPs) in a prototype microfluidic platform is presented. The target molecule recognition is based on volume amplification using padlock-probe-ligation followed by rolling circle amplification (RCA). The MNPs are functionalized with single-stranded oligonucleotides, which give a specific binding of the MNPs to the large RCA coil product, resulting in a large change in the amplitude of the imaginary part of the ac magnetic susceptibility. The RCA products from amplification of synthetic Vibrio cholera target DNA were investigated using our SQUID ac susceptibility system in microfluidic channel with an equivalent sample volume of 3 μl. From extrapolation of the linear dependence of the SQUID signal versus concentration of the RCA coils, it is found that the projected limit of detection for our system is about 1.0 e5 RCA coils (0.2e−18 mol), which is equivalent to 66 fM in the 3 μl sample volume. This ultra-high magnetic sensitivity and integration with microfluidic sample handling are critical steps towards magnetic bioassays for rapid detection of DNA and RNA targets at the point of care.
  •  
40.
  • Shivaji, Kavitha, et al. (författare)
  • Utilization of waste tea leaves as bio-surfactant in CdS quantum dots synthesis and their cytotoxicity effect in breast cancer cells
  • 2019
  • Ingår i: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 487, s. 159-170
  • Tidskriftsartikel (refereegranskat)abstract
    • Green technology for nanoparticles synthesis is considered to be of great significance in biomedical applications. Recently, low dimensional semiconductor cadmium sulfide (CdS) quantum dots (QDs) have raised great attention due to their optical properties and wide usage in biomedical studies. In our present work, we demonstrate a simple green synthesis route for CdS QDs production using waste matured tea leaves (mother leaf) as biosurfactant that are a waste product of the tea leaf industry and not suitable for drinking. The structural and morphological analysis showed waste tea leaf derived CdS QDs range from 2.5 to 4 nm in particle size with a cubic crystalline structure. Interestingly, these CdS QDs exhibit strong fluorescence emission with maximum around 670 nm. We explored the cytotoxic effect of waste tea leaf mediated CdS QDs (MT-CdS QDs) in breast cancer cell lines and compared their viability with standard drug - cisplatin. Our experimental studies strongly suggest that MT-CdS QDs exhibits cytotoxic effect on breast cancer cells and their performance was compared with standard drug cisplatin. To further understand the role of MT-CdS QDs towards cytotoxicity, the fluorescence microscopy and flow cytometry analysis were carried out. The flow cytometry results reveal that MT-CdS QDs induces cell death as it arrests the cell cycle at S phase as well as G2/M phase. Further the apoptosis mechanism was confirmed with the expression of anti-apoptotic and apoptotic proteins. These studies explored that waste tea leaves have dual advantage - both in controlling the particle size of CdS QDs as well as facilitates their cytotoxicity effect in breast cancer cell death. Therefore, it is anticipated that the utilization of MT-CdS QDs produced from waste tea leaves as bi-functional drug and delivery vehicle in cancer treatment will be a promising approach. Also, this is a simple and circular economic route for producing biocompatible QDs at low-cost, which could simultaneously benefit tea and biomedical industries.
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41.
  •  
42.
  • Souza-Moreira, Tatiana M., et al. (författare)
  • Screening of 2A peptides for polycistronic gene expression in yeast
  • 2018
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 18:5
  • Tidskriftsartikel (refereegranskat)abstract
    • A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.
  •  
43.
  • Tiukova, Ievgeniia, 1987, et al. (författare)
  • Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis
  • 2019
  • Ingår i: FEMS microbiology letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 366:17
  • Tidskriftsartikel (refereegranskat)abstract
    • The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.
  •  
44.
  • Ušaj, Marko, et al. (författare)
  • Electrofusion of B16-F1 and CHO cells: the comparison of the pulse first and contact first protocols
  • 2013
  • Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1567-5394 .- 1878-562X. ; 89, s. 34-41
  • Tidskriftsartikel (refereegranskat)abstract
    • High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.
  •  
45.
  • Voinova, Marina, 1958, et al. (författare)
  • Physical processes in polymeric filters used for dialysis
  • 2019
  • Ingår i: Polymers. - : MDPI AG. - 2073-4360. ; 11:3
  • Forskningsöversikt (refereegranskat)abstract
    • The key physical processes in polymeric filters used for the blood purification include transport across the capillary wall and the interaction of blood cells with the polymer membrane surface. Theoretical modeling of membrane transport is an important tool which provides researchers with a quantification of the complex phenomena involved in dialysis. In the paper, we present a dense review of the most successful theoretical approaches to the description of transport across the polymeric membrane wall as well as the cell-polymer surface interaction, and refer to the corresponding experimental methods while studying these phenomena in dialyzing filters.
  •  
46.
  • Westman, Johan, 1983, et al. (författare)
  • A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae
  • 2018
  • Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 6, s. 49-55
  • Tidskriftsartikel (refereegranskat)abstract
    • Yeast flocculation is the reversible formation of multicellular complexes mediated by lectin-like cell wall proteins binding to neighbouring cells. Strong flocculation can improve the inhibitor tolerance and fermentation performance of yeast cells in second generation bioethanol production. The strength of flocculation increases with the size of the flocculation protein and is strain dependent. However, the large number of internal repeats in the sequence of FLO1 from Saccharomyces cerevisiae S288c makes it difficult to recombinantly express the gene to its full length. In the search for novel flocculation genes resulting in strong flocculation, we discovered a DNA sequence, FLONF, that gives NewFlo phenotype flocculation in S. cerevisiae CEN.PK 113-7D. The nucleotide sequence of the internal repeats of FLONF differed from those of FLO1. We hypothesized that a chimaeric flocculation gene made up of a FLO1 variant derived from S. cerevisiae S288c and additional repeats from FLONF from S. cerevisiae CCUG 53310 would be more stable and easier to amplify by PCR. The constructed gene, FLOw, had 22 internal repeats compared to 18 in FLO1. Expression of FLOw in otherwise non-flocculating strains led to strong flocculation. Despite the length of the gene, the cassette containing FLOw could be easily amplified and transformed into yeast strains of different genetic background, leading to strong flocculation in all cases tested. The developed gene can be used as a self-immobilization technique or to obtain rapidly sedimenting cells for application in e.g. sequential batches without need for centrifugation.
  •  
47.
  • Yu, R., et al. (författare)
  • Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast
  • 2020
  • Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwisereducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining. © 2020, The Author(s).
  •  
48.
  • Zou, Gen, et al. (författare)
  • Harnessing synthetic biology for mushroom farming
  • 2023
  • Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 41:4, s. 480-483
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • Recent advances in synthetic biology have transformed mushroom farming from a focus on traditional cultivation to comprehensive applications based on cutting-edge biotechnologies. Synthetic biology has promising applications in this field, including precision breeding, mining biosynthetic gene clusters, developing mushroom chassis cells, and constructing cell factories for high value-added products.
  •  
49.
  • Apelgren, Peter, et al. (författare)
  • Biomaterial and biocompatibility evaluation of tunicate nanocellulose for tissue engineering.
  • 2022
  • Ingår i: Biomaterials advances. - : Elsevier BV. - 2772-9508. ; 137
  • Tidskriftsartikel (refereegranskat)abstract
    • Extracellular matrix fibril components, such as collagen, are crucial for the structural properties of several tissues and organs. Tunicate-derived cellulose nanofibrils (TNC) combined with living cells could become the next gold standard for cartilage and soft-tissue repair, as TNC fibrils present similar dimensions to collagen, feasible industrial production, and chemically straightforward and cost-efficient extraction procedures. In this study, we characterized the physical properties of TNC derived from aquaculture production in Norwegian fjords and evaluated its biocompatibility regarding induction of an inflammatory response and foreign-body reactions in a Wistar rat model. Additionally, histologic and immunohistochemical analyses were performed for comparison with expanded polytetrafluoroethylene (ePTFE) as a control. The average length of the TNC as determined by atomic force microscopy was tunable from 3μm to 2.4μm via selection of a various number of passages through a microfluidizer, and rheologic analysis showed that the TNC hydrogels were highly shear-thinning and with a viscosity dependent on fibril length and concentration. As a bioink, TNC exhibited excellent rheological and printability properties, with constructs capable of being printed with high resolution and fidelity. We found that post-print cross-linking with alginate stabilized the construct shape and texture, which increased its ease of handling during surgery. Moreover, after 30days in vivo, the constructs showed a highly-preserved shape and fidelity of the grid holes, with these characteristics preserved after 90days and with no signs of necrosis, infection, acute inflammation, invasion of neutrophil granulocytes, or extensive fibrosis. Furthermore, we observed a moderate foreign-body reaction involving macrophages, lymphocytes, and giant cells in both the TNC constructs and PTFE controls, although TNC was considered a non-irritant biomaterial according to ISO 10993-6 as compared with ePTFE. These findings represent a milestone for future clinical application of TNC scaffolds for tissue repair. One sentence summary: In this study, the mechanical properties of tunicate nanocellulose are superior to nanocellulose extracted from other sources, and the biocompatibility is comparable to that of ePTFE.
  •  
50.
  • Brunius, Carl, 1974, et al. (författare)
  • Prediction and modeling of pre-analytical sampling errors as a strategy to improve plasma NMR metabolomics data
  • 2017
  • Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1460-2059 .- 1367-4811. ; 33:22, s. 3567-3574
  • Tidskriftsartikel (refereegranskat)abstract
    • Biobanks are important infrastructures for life science research. Optimal sample handling regarding e.g. collection and processing of biological samples is highly complex, with many variables that could alter sample integrity and even more complex when considering multiple study centers or using legacy samples with limited documentation on sample management. Novel means to understand and take into account such variability would enable high-quality research on archived samples. This study investigated whether pre-analytical sample variability could be predicted and reduced by modeling alterations in the plasma metabolome, measured by NMR, as a function of pre-centrifugation conditions (1-36 h pre-centrifugation delay time at 4 A degrees C and 22 A degrees C) in 16 individuals. Pre-centrifugation temperature and delay times were predicted using random forest modeling and performance was validated on independent samples. Alterations in the metabolome were modeled at each temperature using a cluster-based approach, revealing reproducible effects of delay time on energy metabolism intermediates at both temperatures, but more pronounced at 22 A degrees C. Moreover, pre-centrifugation delay at 4 A degrees C resulted in large, specific variability at 3 h, predominantly of lipids. Pre-analytical sample handling error correction resulted in significant improvement of data quality, particularly at 22 A degrees C. This approach offers the possibility to predict pre-centrifugation delay temperature and time in biobanked samples before use in costly downstream applications. Moreover, the results suggest potential to decrease the impact of undesired, delay-induced variability. However, these findings need to be validated in multiple, large sample sets and with analytical techniques covering a wider range of the metabolome, such as LC-MS.
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