| 1. |
- De Oliveira, Danilo Hirabae
(författare)
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Integrative structural biology of protein fibers: Spider silk and beta-lactoglobulin nanofibrils
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins found in nature offer a vast range of exceptional materials, including high-performancebiopolymers such as spider silks and whey protein nanofibrils. Fibrous proteins possess immensepotential for developing novel materials suited for various applications, such as medicalbiomaterials or industrial products. This thesis uses an interdisciplinary approach based onexperimental and computational methods to present insights into the fundamental aspects ofprotein fibers, exploring details on the molecular level and their self-assembly.Spider silk threads exhibit strength, elasticity, and the ability to withstand high-energy loads.Additionally, silk is naturally degradable, and compatible with cell growth, and non-immunogenic.This thesis examines the molecular assembly of spider silk, particularly the secondary structurelevel, which contributes greatly to its properties. We unveil the structural details of therecombinant spidroins 4RepCT and the CT domain over hydrophobic/hydrophilic surfaces,describing their periodic oriented macrostructure and stability. Furthermore, it is reported that theCT domain form β-nanocrystalline components, revealing a specific segment (helix No4) that canself-assemble into nanofibrils in a pH-sensitive manner. In addition, we describe the method ofsortase-mediated transpeptidation reaction used to catalyze the covalent coupling of the spidroins4Rep and CT, resulting in partially isotopically labeled fibers suitable for solid-state NMRspectroscopy analyses.β-lactoglobulin is an emerging protein source used to create advanced biomaterials because of itshigh availability and ability to assemble to protein nanofibrils (PNFs). Recombinant and syntheticβ-lactoglobulin PNFs with isotopic labelling are generated and analyzed using solid-state NMRspectroscopy and atomic force microscopy. The fibrils of both species present congruenciesregarding morphology with unbranched conformation and a height of approximately 6 nm. At thesame time, their NMR spectra demonstrate accordance with their hydrophobic residues (i.e., Ala,Val, Ile, and Leu) as β-sheets. In addition, distinct inter-residue cross-peaks of Ser-Thr and LeuIle provide insights into the molecular structure of β-lactoglobulin PNF.This thesis presents new knowledge about the hierarchy of protein fibrils and the structure ofprotein-based materials at the molecular level. This knowledge can unlock the design anddevelopment of innovative protein-based materials for various applications.
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| 2. |
- Dillner, Joakim, et al.
(författare)
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Antibodies to SARS-CoV-2 and risk of past or future sick leave
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The extent that antibodies to SARS-CoV-2 may protect against future virus-associated disease is unknown. We invited all employees (n=15,300) at work at the Karolinska University Hospital, Stockholm, Sweden to participate in a study examining SARS-Cov-2 antibodies in relation to registered sick leave. For consenting 12,928 healthy hospital employees antibodies to SARS-CoV-2 could be determined and compared to participant sick leave records. Subjects with viral serum antibodies were not at excess risk for future sick leave (adjusted odds ratio (OR) controlling for age and sex: 0.85 [95% confidence interval (CI) (0.85 (0.43-1.68)]. By contrast, subjects with antibodies had an excess risk for sick leave in the weeks prior to testing [adjusted OR in multivariate analysis: 3.34 (2.98-3.74)]. Thus, presence of viral antibodies marks past disease and protection against excess risk of future disease. Knowledge of whether exposed subjects have had disease in the past or are at risk for future disease is essential for planning of control measures.Trial registration: First registered on 02/06/20, ClinicalTrials.gov NCT04411576.
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| 3. |
- Havervall, Sebastian, et al.
(författare)
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Robust humoral and cellular immune responses and low risk for reinfection at least 8 months following asymptomatic to mild COVID-19
- 2022
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Ingår i: Journal of Internal Medicine. - : John Wiley & Sons. - 0954-6820 .- 1365-2796. ; 291:1, s. 72-80
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Tidskriftsartikel (refereegranskat)abstract
- Background: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts.Methods: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points.Results: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%).Conclusions: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.
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| 4. |
- Havervall, Sebastian, et al.
(författare)
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SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection
- 2022
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 17:1, s. e0262169-e0262169
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Tidskriftsartikel (refereegranskat)abstract
- Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10−23 and 2*10−13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys
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| 5. |
- Hedhammar, My, Professor, 1975-, et al.
(författare)
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Zbasic : A Purification Tag for Selective Ion-Exchange Recovery
- 2021
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Ingår i: Protein Downstream Processing. - New York, NY : Humana Press. ; , s. 149-158
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Bokkapitel (refereegranskat)abstract
- A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein. Herein, protocols for purification of soluble Zbasic-tagged fusion proteins, as well as for integrated purification and solid-phase refolding of insoluble fusion proteins, are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.
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| 6. |
- Hober, Sophia, Professor, 1965-, et al.
(författare)
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Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay
- 2021
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Ingår i: Clinical & Translational Immunology. - : Wiley. - 2050-0068. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Objective. The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. Methods. More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. Results. Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. Conclusion. These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.
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| 7. |
- Lauren, Ida, et al.
(författare)
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Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology
- 2022
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Ingår i: Immunity, Inflammation and Disease. - : John Wiley & Sons. - 2050-4527. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. Methods: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFN gamma) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. Results: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFN gamma and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFN gamma in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. Conclusion: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.
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| 8. |
- Mravinacová, Sára, et al.
(författare)
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A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies
- 2022
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 66, s. 46-52
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Tidskriftsartikel (refereegranskat)abstract
- Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.
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| 9. |
- Ornithopoulou, Eirini
(författare)
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Hierarchical Assembly Investigations and Multiscale Characterization of Protein-based Materials : Insights from Whey Protein Nanofibrils and Recombinant Spider Silk Microspheres
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-based materials, with their unique properties of combining high strength, while maintaining elasticity, and their inherent biocompatibility, hold immense potential for various applications. In order to harness these properties, we need to understand and control how protein building blocks come together to form hierarchically structured materials. Using critical thinking when combining different proteins may lead to advanced materials with synergistic effects that can tackle complex problems such as targeted drug delivery. This thesis presents an investigation into the behavior of some protein-based materials, specifically whey protein isolate nanofibrils, β-lactoglobulin peptide fragment assemblies, and recombinant spider silk microspheres.In Paper I, the hierarchical assembly and packing behavior of whey protein nanofibrils were in situ investigated using a Liquid Bridge Induced Assembly setup and X-ray Scattering, along with Atomic Force Microscopy and Scanning Electron Microscopy. The results demonstrated that the alignment of straight and curved fibrils was affected by temperature and fibril size, providing insights into assembly dynamics for future material production.In Paper II, the impact of nanoscale features of whey protein nanofibrils on the morphology of films was investigated. It was found that controlling fibril size and employing fast-drying protocols could manipulate macroscale features without sacrificing the functional properties of the nanofibrils.In Paper III, the nanoscale morphology, molecular arrangement, and polymorphism of protein nanofibrils formed by a synthetic peptide fragment derived from β-lactoglobulin were examined. Β-lactoglobulin is the only nanofibril forming component in whey protein isolate. Results suggested that polymorphism stems from protofilament packing differences at the nanoscale, and a possible parallel steric zipper packing.In Paper IV, the self-assembly behavior of a recombinant spider silk protein in physiological like buffer and with the addition of hyaluronic acid was explored. The self-assembled FN-silk microspheres demonstrated a fibrillar or porous mesostructure. 2D and 3D cell culture trials show that the microspheres could have potential applications in biomedicine.Taken together, the acquired knowledge will contribute to our fundamental understanding of protein-based materials, especially those similar to PNF-based and recombinant spider silk-based materials and inform the design of improved and innovative materials in biomanufacturing, such as functional textiles and surface biofunctionalization.
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| 10. |
- Tasiopoulos, Christos Panagiotis, 1985-
(författare)
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Recombinant spider silk for biomedical applications - from functionalizing surfaces of synthetic materials to in vitro modelling of tissues
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Spider silk is a natural protein-based material known for its medicinal use and remarkable mechanical properties. Structures made thereof are both strong and elastic and have been shown to be favorable matrices for tissue engineering. As natural spider silk is difficult to obtain, recombinant technology is instead used to produce partial silk proteins. This thesis investigates the use of one such partial spider silk protein functionalized with a cell adhesion motif from fibronectin, FN-4RepCT, to coat the surface of synthetic polymers, as well as self-assemble into nanofibrillar membranes for modelling of tissues in vitro.In Paper I, the silk protein was shown to self-assemble into coatings with simultaneous entrapment of cells (co-seeding) to functionalize polymeric surfaces. The results showed that the co-seeding approach facilitated the adherence and sustained the viability of cells on surfaces of materials widely used for the manufacture of cardiovascular grafts.In Paper II, a protocol was developed to enable the formation of nanofibrillar coatings on the surface of membranes intended for guided bone regeneration. This was done by reducing the surface tension of the membranes, allowing for the self-assembly of silk proteins to take place. The silk coating facilitated the adherence, promoted the growth, and mediated the generation of a cell monolayer of tissue representative cells seeded on either side of the membrane.In Paper III, the self-assembly of the silk protein at the air-liquid interface was shown to form cm-sized free-standing, tough and elastic, nanofibrillar silk nanomembranes, permeable to macromolecules of various sizes, and able to support the establishment of a confluent layer of keratinocytes seeded on either side. In Paper IV, the nanofibrillar silk membranes were shown able to support cell co-culture to generate a model of the blood vessel wall in vitro.In Paper V, an alveolar-capillary model was established by seeding lung epithelial and endothelial cells on opposite sides of nanofibrillar silk membranes. The results showed the formation of an in vivo like tissue through the expression of junctional complexes and the production of essential surfactants. The silk membranes were also for the first time integrated into a microfluidic device to expose the endothelium to flow-induced shear stresses.Altogether, the work conducted in this thesis shows promise to the use of the FN-4RepCT silk protein both for coating surfaces of bio-inert synthetic polymeric materials and forming thin and nanofibrillar membranes for the engineering of tissues in vitro.
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