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  • 1.
    Abdel-Rehim, Mohamed
    Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences. AstraZeneca R&D Sodertalje, Global DMPK, SE-15185 Sodertalje, Sweden.;Stockholm Univ, Dept Analyt Chem, SE-10691 Stockholm, Sweden.;Karlstad Univ, Dept Chem & Biomed Sci, Fac Sci & Technol, SE-65188 Karlstad, Sweden..
    Microextraction by packed sorbent (MEPS): A tutorial2011In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 701, no 2, p. 119-128Article in journal (Refereed)
    Abstract [en]

    This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided. In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed. (C) 2011 Elsevier B.V. All rights reserved.

  • 2.
    Abdel-Rehim, Mohamed
    Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences. AstraZeneca R&D Sodertalje, Global DMPK, Sodertalje, Sweden.;Karlstad Univ, Fac Sci & Technol, Dept Chem & Biomed Sci, Karlstad, Sweden..
    On-Line Whole Blood Analysis Using Microextraction by Packed Sorbent and LC-MS-MS2011In: LC GC North America, ISSN 1527-5949, E-ISSN 1939-1889, Vol. 29, no 7, p. 612-618Article in journal (Refereed)
    Abstract [en]

    Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with liquid chromatography (LC) or gas chromatography (GC) systems without any modifications. This article describes the use of MEPS in clinical and preclinical studies to quantify different drugs in whole blood samples. MEPS was used to determine cyclophosphamide in mouse blood from preclinical g studies using 20 mu L of blood samples. The interday accuracies and 0 precisions ranged from 107-109% and from 2.0-7.0%, respectively. The determination of four immunosuppressive drugs in human blood by MEPS and liquid chromatography-mass spectrometry (LC-MS) is described. The method showed a good selectivity and sensitivity. The calibration curves for everolimus, sirolimus, and tacrolimus ranged from 0.5 to 50 ng/mL and for cyclosporine from 3.0 to 1500 ng/mL. Intraday precisions for the studied immunosuppressive drugs were 2.0-11.7% and interday precision ranged from 5.1 to 13.7% (CV).

  • 3.
    Abdiwoli, Abdisalam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    pH-responsive release of proteins from colloidal capsules for oral drug delivery2020Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Biologics are an important part of modern healthcare and are mostly administered parenterally due to the fact that it is the route of administration that avoids degradation of biologics and ensures their systemic exposure. However, there is a need to develop oral drug delivery formulations for local treatment of diseases in the gastrointestinal tract (GI). Colloidal capsules is a formulation that can potentially facilitate oral administration of biologics. There have been studies on colloidal capsules and the various ways of manufacturing them, one of which is “Emulsion-based method”. The aim of this study was to produce colloidal capsules made of silica nanoparticles through emulsion-based method, coat them to study their pH-responsive release and characterize them. Encapsulation of a model protein in the silica colloidal capsules was also attempted. pH-responsive release was not studied due to limited access to the laboratory and, a literature study of articles about colloidal capsules was conducted instead, regarding different aspects of colloidal capsule synthesis and encapsulation of various compunds. Web of science was the database used to find scientific studies that specifically produced colloidal capsules. Colloidal capsules were synthesized using a Pickering-emulsion method. Commercially available SiO2 nanoparticles were used to form the capsules by ultrasonication.  The hydrodynamic size and capsule morphology were analyzed using dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively. Zeta potential was measured through electrophoretic light scattering (ELS). Articles for the literature study were found using the “web of science” database. Colloidal capsules were successfully produced, coated and characterized. Additionally, the literature study shows that there diverse colloidal capsule synthesis conditions, model proteins and applications for colloidal capsules.

  • 4.
    Abouzayed, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Seitova, Kamila
    Siberian State Med Univ, Sci & Res Lab Chem & Pharmaceut Res, Tomsk, Russia.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk, Russia..
    Lundmark, Fanny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Bodenko, Vitalina
    Siberian State Med Univ, Sci & Res Lab Chem & Pharmaceut Res, Tomsk, Russia.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk, Russia..
    Oroujeni, Maryam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Affibody AB, Solna, Sweden..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk, Russia..
    Rosenström, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    177Lu-labeled PSMA targeting therapeutic with optimized linker for treatment of disseminated prostate cancer; evaluation of biodistribution and dosimetry2023In: Frontiers in Oncology, E-ISSN 2234-943X, Vol. 13, article id 1221103Article in journal (Refereed)
    Abstract [en]

    <bold>Introduction:</bold> Prostate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMA-expression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that Lu-177-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy.<bold>Methods:</bold> BQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [Lu-177]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [Lu-177]Lu-PSMA-617 was simultaneously evaluated for comparison.<bold>Results:</bold> BQ7876 was labeled with Lu-177 with radiochemical yield >99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [Lu-177]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities K-D1 = 3.8 nM and K-D2 = 25 nM. The half-maximal inhibitory concentration for Lu-nat-BQ7876 was 59 nM that is equal to 61 nM for Lu-nat-PSMA-617. Cellular processing of [Lu-177]Lu-BQ7876 was accompanied by slow internalization. [Lu-177]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 +/- 3%ID/g) did not differ significantly from tumor uptake (9 +/- 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen.<bold>Discussion:</bold> Biodistribution studies in mice demonstrated that targeting properties of [Lu-177]Lu-BQ7876 are not inferior to properties of [Lu-177]Lu-PSMA-617, but do not offer any decisive advantages.

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  • 5.
    Abrahamsson, Annelie
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
    Rasti Boroojeni, Fatemeh
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering.
    Naeimipour, Sajjad
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering.
    Reustle, Nina
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering.
    Selegård, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Biophysics and bioengineering. Linköping University, Faculty of Science & Engineering.
    Dabrosin, Charlotta
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Increased matrix stiffness enhances pro-tumorigenic traits in a physiologically relevant breast tissue- monocyte 3D model2024In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 178, p. 160-169Article in journal (Refereed)
    Abstract [en]

    High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se . In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro -inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross -linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer.

  • 6. Abramson, Alex
    et al.
    Frederiksen, Morten Revsgaard
    Vegge, Andreas
    Jensen, Brian
    Poulsen, Mette
    Mouridsen, Brian
    Jespersen, Mikkel Oliver
    Kirk, Rikke Kaae
    Windum, Jesper
    Hubálek, František
    Water, Jorrit J.
    Fels, Johannes
    Gunnarsson, Stefán B.
    Bohr, Adam
    Straarup, Ellen Marie
    Ley, Mikkel Wennemoes Hvitfeld
    Lu, Xiaoya
    Wainer, Jacob
    Collins, Joy
    Tamang, Siddartha
    Ishida, Keiko
    Hayward, Alison
    Herskind, Peter
    Buckley, Stephen T.
    Roxhed, Niclas
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems. MIT, Dept Chem Engn, 77 Massachusetts Ave, Cambridge, MA 02139 USA; MIT, David H Koch Inst Integrat Canc Res, 77 Massachusetts Ave, Cambridge, MA 02139 USA.
    Langer, Robert
    Rahbek, Ulrik
    Traverso, Giovanni
    Oral delivery of systemic monoclonal antibodies, peptides and small molecules using gastric auto-injectors2021In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 40, no 1, p. 103-109Article in journal (Refereed)
    Abstract [en]

    Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system. 

  • 7.
    Abualreesh, Heba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Screening for antibacterial metabolites in marine sponges collected from the coastline of Sri Lanka.2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Natural products and their derivatives have and are still used by humans for various health ailments due to their rich sources of drug discovery. New biologically active compounds from natural products play a key role in drug development. Marine sponges and their associated microbes contain a lot of bioactive compounds that are potential for drug development. These compounds produce chemical compounds with useful pharmaceutical properties such as antitumor, anti-infective, anti-inflammatory, and antibacterial properties. The main focus of this project was on the antibacterial activity of six different sponge specimens. The aim was to screen the antibacterial activity of the sponge specimen’s extracts. In order to do so, a Minimum Inhibitory Concentration assay was performed to screen the sponge's antibacterial activity against E. coli and S. aureus. Analytical HPLC was used for separation and Solid Phase Extraction (SPE) was used for determining the effect of salts towards the inhibition of anti-bacterial activity for two selected extracts. Ethanolic extract of Stylissa massa showed antibacterial activity against S. aureus. SPE would be a rapid purification step to remove the salts present in sponges at a high concentration but it has not shown a significant effect on the inhibition of antibacterial activity. However, further separation and purification need to be done to be able to completely screen for all the six different sponge specimens.

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  • 8. Acero Sanchez, Josep Ll.
    et al.
    Joda, Hamdi
    Henry, Olivier Y. F.
    Solnestam, Beata W.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laddach, Nadja
    Ramakrishnan, Dheeraj
    Riley, Ian
    Schwind, Carmen
    Latta, Daniel
    O'Sullivan, Ciara K.
    Electrochemical Genetic Profiling of Single Cancer Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3378-3385Article in journal (Refereed)
    Abstract [en]

    Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

  • 9.
    Acharya, Shikha
    et al.
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    Jin, Chunsheng
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Med Biochem & Cell Biol, Gothenburg, Sweden.
    Bylund, Johan
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    Shen, Qiujin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jontell, Mats
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Med & Pathol, Gothenburg, Sweden.
    Carlen, Anette
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    Karlsson, Niclas G.
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Med Biochem & Cell Biol, Gothenburg, Sweden.
    Reduced sialyl-Lewis(x) on salivary MUC7 from patients with burning mouth syndrome2019In: Molecular Omics, E-ISSN 2515-4184, Vol. 15, no 5, p. 331-339Article in journal (Refereed)
    Abstract [en]

    We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewis(x) (Si-Le(x)) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewis(x) (Si-Le(x)) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Le(x), may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Le(x) remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.

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  • 10. Addario, Barbara
    et al.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Backman, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Characterisation of Schizosaccharomyces pombe alpha-actinin2016In: PeerJ, E-ISSN 2167-8359, Vol. 4, article id e1858Article in journal (Refereed)
    Abstract [en]

    The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin.

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  • 11.
    Adhikari, Deepak
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Flohr, Gilian
    Hogeschool Leiden, Zernikedreef 11,2333 CK Leiden, The Netherlands.
    Gorre, Nagaraju
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yang, Hairu
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lan, Zijian
    University of Louisville Health Sciences Center, Louisville, Kentucky, USA.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles2009In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 15, no 12, p. 765-770Article in journal (Refereed)
    Abstract [en]

    To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1-Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

  • 12.
    Adil, Nurmeen
    et al.
    Univ Karachi, HEJ Res Inst Chem, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    Ali, Hamad
    Univ Karachi, Dr Panjwani Ctr Mol Med & Drug Res, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    Siddiqui, Amna Jabbar
    Univ Karachi, Dr Panjwani Ctr Mol Med & Drug Res, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    Ali, Arslan
    Univ Karachi, Dr Panjwani Ctr Mol Med & Drug Res, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    Ahmed, Ayaz
    Univ Karachi, Dr Panjwani Ctr Mol Med & Drug Res, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    El-Seedi, Hesham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Jiangsu Univ, Int Res Ctr Food Nutr & Safety, Zhenjiang 212013, Jiangsu, Peoples R China..
    Musharraf, Syed Ghulam
    Univ Karachi, HEJ Res Inst Chem, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan.;Univ Karachi, Dr Panjwani Ctr Mol Med & Drug Res, Int Ctr Chem & Biol Sci, Karachi 75270, Pakistan..
    Evaluation of cytotoxicity of areca nut and its commercial products on normal human gingival fibroblast and oral squamous cell carcinoma cell lines2021In: Journal of Hazardous Materials, ISSN 0304-3894, E-ISSN 1873-3336, Vol. 403, article id 123872Article in journal (Refereed)
    Abstract [en]

    Consumption of areca nut products is the most common cause of oral cancers, particularly in South Asian countries. This study evaluates the cytotoxic and necrotizing effects of areca nut and its formulations on normal human gingival fibroblasts (HGF-1) and oral squamous cell carcinoma (OSCC, CAL-27) cell lines. Identification of various carcinogens and adulterants using LC-HR-ESI-MS/MS analysis was performed in the extracts of areca nut and its products. Apart from alkaloids and flavonoids, a major adulterant, saccharin was found in all the samples of chalia (one of the most common chewing products of areca nut) in the ranges between 1.697-7.170 mg/g of the sample. Cytotoxic studies showed that most of the areca nut products were found cytotoxic to HGF-1 cells while being relatively non-cytotoxic against CAL-27 cells, rather they promote the growth of cancer cells. Our findings revealed that the components of areca nut and its products were injurious to HGF-1 cells and caused necrosis, which may attenuate HGF-1 protection toward oral epithelial cells. Moreover, the non-cytotoxic effect of these products on cancer cell lines suggests further predisposal of the habitual chewers for developing oral carcinomas. This study will give a better understanding of the hazardous effects of areca nut products.

  • 13.
    Adrian-Kalchhauser, Irene
    et al.
    Univ Basel, Program Man Soc Environm, Dept Environm Sci, Vesalgasse 1, CH-4051 Basel, Switzerland..
    Svensson, Ola
    Univ Gothenburg, Dept Biol & Environm Sci, Medicinaregatan 18A, S-41390 Gothenburg, Sweden.;Univ Gothenburg, Linnaeus Ctr Marine Evolutionary Biol, POB 46040530, Gothenburg, Sweden..
    Kutschera, Verena E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Rosenblad, Magnus Alm
    Univ Gothenburg, Linnaeus Ctr Marine Evolutionary Biol, POB 46040530, Gothenburg, Sweden.;Univ Gothenburg, Dept Marine Sci, NBIS Bioinformat Infrastruct Life Sci, Medicinaregatan 9C, S-41390 Gothenburg, Sweden..
    Pippel, Martin
    Heidelberg Inst Theoret Studies, Schloss Wolfsbrunnenweg 35, D-69118 Heidelberg, Germany..
    Winkler, Sylke
    Max Planck Inst Mol Cell Biol & Genet, Pfotenhauerstr 108, D-01307 Dresden, Germany..
    Schloissnig, Siegfried
    Heidelberg Inst Theoret Studies, Schloss Wolfsbrunnenweg 35, D-69118 Heidelberg, Germany..
    Blomberg, Anders
    Univ Gothenburg, Linnaeus Ctr Marine Evolutionary Biol, POB 46040530, Gothenburg, Sweden.;Univ Gothenburg, Dept Marine Sci, Medicinaregatan 9C, S-41390 Gothenburg, Sweden..
    Burkhardt-Holm, Patricia
    Univ Basel, Program Man Soc Environm, Dept Environm Sci, Vesalgasse 1, CH-4051 Basel, Switzerland.;Univ Alberta, Dept Biol Sci, 11455 Saskatchewan Dr, Edmonton, AB, Canada..
    The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish2017In: BMC Genomics, E-ISSN 1471-2164, Vol. 18, article id 177Article in journal (Refereed)
    Abstract [en]

    Background: Vertebrate mitochondrial genomes are optimized for fast replication and low cost of RNA expression. Accordingly, they are devoid of introns, are transcribed as polycistrons and contain very little intergenic sequences. Usually, vertebrate mitochondrial genomes measure between 16.5 and 17 kilobases ( kb). Results: During genome sequencing projects for two novel vertebrate models, the invasive round goby and the sand goby, we found that the sand goby genome is exceptionally small (16.4 kb), while the mitochondrial genome of the round goby is much larger than expected for a vertebrate. It is 19 kb in size and is thus one of the largest fish and even vertebrate mitochondrial genomes known to date. The expansion is attributable to a sequence insertion downstream of the putative transcriptional start site. This insertion carries traces of repeats from the control region, but is mostly novel. To get more information about this phenomenon, we gathered all available mitochondrial genomes of Gobiidae and of nine gobioid species, performed phylogenetic analyses, analysed gene arrangements, and compared gobiid mitochondrial genome sizes, ecological information and other species characteristics with respect to the mitochondrial phylogeny. This allowed us amongst others to identify a unique arrangement of tRNAs among Ponto-Caspian gobies. Conclusions: Our results indicate that the round goby mitochondrial genome may contain novel features. Since mitochondrial genome organisation is tightly linked to energy metabolism, these features may be linked to its invasion success. Also, the unique tRNA arrangement among Ponto- Caspian gobies may be helpful in studying the evolution of this highly adaptive and invasive species group. Finally, we find that the phylogeny of gobiids can be further refined by the use of longer stretches of linked DNA sequence.

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  • 14.
    Afify, Khaldon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Recombinant expression of; a precursor of cyclotide kalata S, a cyclic dimer of the antimicrobial peptide KR-12 and a mushroom-derived peptide2020Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Abstract The biological activities of the living organisms are performed and controlled by various kinds of functional proteins. A lot of studies are being done to unveil the secrets of the multiple functions of proteins. Protein functional studies often require pure and large quantities, however, this is hampered by small concentrations produced naturally in human, plants or animal tissue. Cyclotides are an example of proteins that exist on tiny amounts in plants. Recombinant protein expression is an alternative solution to produce proteins of interest on large scales. Furthermore, recombinant protein expression can be exploited to produce chemically synthesized or structurally modified proteins derived from naturally occurring proteins. Novel modified antimicrobial peptides are an example of proteins that are being developed in labs. Here, we utilized E. coli to express a series of different medicinal peptides: a full precursor of cyclotide kalata S from plant Viola Odorata, the cyclic dimer derived from the antimicrobial peptide LL-37 and a peptide produced by Kusaghizi mushroom that grows naturally in Tanzania. Our results showed a successful recombinant expression in E. coli for, the precursor of cyclotide kalata S and the different fragments of it, the backbone of a derived variant of the antimicrobial peptide LL-37 and the mushroom-derived peptide.

  • 15.
    Aggarwal, Tanya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Patil, Sourabh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ceder, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hayder, Maher
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Knockdown of SLC38 Transporter Ortholog-CG13743 Reveals a Metabolic Relevance in Drosophila2020In: Frontiers in Physiology, E-ISSN 1664-042X, Vol. 10, article id 1592Article in journal (Refereed)
    Abstract [en]

    Solute Carrier (SLC) is a cluster of families of membrane bound transporters, of which many members lack defined substrate profile, and many more are poorly characterized. Many play a vital role in regulating metabolic systems, protein synthesis, and post translational modifications. SLC38 is one of the families of SLCs, which are also known as sodium-coupled neutral amino acid transporters (SNATs). In mice, it has 11 members (SNAT1-11) but in Drosophila there are two homologs for the SLC38 family; CG13743 and CG30394. Here, we show characteristics of Drosophila CG13743 which closely resembles SLC38A11 in humans. SLC38A11 still remains an orphan member of the SLC38 family which has not been functionally well studied. We used the UAS-GAL4 system to investigate and control gene expression using RNAi lines for ubiquitous knockdown of the CG13743 gene. It was found to be expressed mainly in salivary gland and brain. Knockdown flies had reduced body weight and consumed less sugar compared with controls. The gene knockdown also affected stored energy pools (lipids and glycogen) and influenced feeding pattern and total activity. In all, this shows novel findings for the characterization of CG13743 in Drosophila and a possible role in maintaining general metabolic pathways and behavior of the fly.

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  • 16.
    Aguilar-Calvo, Patricia
    et al.
    Univ Calif San Diego, CA 92093 USA; Pfizer, CA USA.
    Malik, Adela
    Univ Calif San Diego, CA 92093 USA; Fate Therapeut, CA USA.
    Sandoval, Daniel R.
    Univ Calif San Diego, CA USA; GSK plc, PA USA.
    Barback, Christopher
    Univ Calif San Diego, CA USA; Univ Colorado, CO USA.
    Orru, Christina D.
    NIAID, MT USA.
    Standke, Heidi G.
    Case Western Reserve Univ, OH USA.
    Thomas, Olivia R.
    Case Western Reserve Univ, OH USA.
    Dwyer, Chrissa A.
    Univ Calif San Diego, CA USA.
    Pizzo, Donald P.
    Univ Calif San Diego, CA 92093 USA.
    Bapat, Jaidev
    Univ Calif San Diego, CA 92093 USA.
    Soldau, Katrin
    Univ Calif San Diego, CA 92093 USA.
    Ogawa, Ryotaro
    Univ Calif San Diego, CA USA.
    Riley, Mckenzie B.
    Univ Alabama Birmingham, AL USA.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Kraus, Allison
    Case Western Reserve Univ, OH USA.
    Caughey, Byron
    NIAID, MT USA.
    Iliff, Jeffrey J.
    VA Puget Sound Hlth Care Syst, WA USA; Univ Washington, WA USA.
    Vera, David R.
    Univ Calif San Diego, CA USA.
    Esko, Jeffrey D.
    Univ Calif San Diego, CA USA.
    Sigurdson, Christina J.
    Univ Calif San Diego, CA 92093 USA; UC San Diego Hlth, CA 92093 USA; Bristol Myers Squibb, CA USA.
    Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection2023In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 19, no 9, article id e1011487Article in journal (Refereed)
    Abstract [en]

    Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a major extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase), from neurons or astrocytes, we then investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, only affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of mice expressing unsulfated HS, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance. Prions cause a rapidly progressive neurologic disease and death with no curative treatment available. Prion aggregates accumulate exponentially in the brain of affected individuals triggering neuronal loss and neuroinflammation, yet the molecules that facilitate prion protein aggregation are largely unknown. We have found that prions in the brain preferentially bind to a highly sulfated endogenous polysaccharide, known as heparan sulfate (HS). Here we use genetically modified mice that express poorly sulfated, neuron-derived HS, and infect mice with different prions strains. We find that mice infected with a plaque-forming prion strain show a prolonged survival and fewer plaques compared to controls. We also found that recombinant prion protein was efficiently transported within the interstitial fluid of mice having poorly sulfated HS, suggesting more efficient clearance from the brain. Our study provides insight into how HS retains prion aggregates in the brain to accelerate disease and indicates a specific HS biosynthetic enzyme to target to enhance protein clearance.

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  • 17.
    Aguilar-Calvo, Patricia
    et al.
    Univ Calif, CA 92093 USA.
    Sevillano, Alejandro M.
    Univ Calif, CA 92093 USA.
    Bapat, Jaidev
    Univ Calif, CA 92093 USA.
    Soldau, Katrin
    Univ Calif, CA 92093 USA.
    Sandoval, Daniel R.
    Univ Calif, CA USA.
    Altmeppen, Hermann C.
    Univ Med Ctr Hamburg, Germany.
    Linsenmeier, Luise
    Univ Med Ctr Hamburg, Germany.
    Pizzo, Donald P.
    Univ Calif, CA 92093 USA.
    Geschwind, Michael D.
    Univ Calif San Francisco, CA USA.
    Sanchez, Henry
    Univ Calif San Francisco, CA USA.
    Appleby, Brian S.
    Case w Reserve Univ, OH USA.
    Cohen, Mark L.
    Case w Reserve Univ, OH USA.
    Safar, Jiri G.
    Case w Reserve Univ, OH USA.
    Edland, Steven D.
    Univ Calif, CA USA.
    Glatzel, Markus
    Univ Med Ctr Hamburg, Germany.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Esko, Jeffrey D.
    Univ Calif, CA USA.
    Sigurdson, Christina J.
    Univ Calif, CA USA.
    Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions2020In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 139, no 3, p. 527-546Article in journal (Refereed)
    Abstract [en]

    Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1(+/-)) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

  • 18.
    Aguilo, Francesca
    et al.
    Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
    Zakirova, Zuchra
    Nolan, Katie
    Wagner, Ryan
    Sharma, Rajal
    Hogan, Megan
    Wei, Chengguo
    Sun, Yifei
    Walsh, Martin J.
    Kelley, Kevin
    Zhang, Weijia
    Ozelius, Laurie J.
    Gonzalez-Alegre, Pedro
    Zwaka, Thomas P.
    Ehrlich, Michelle E.
    THAP1: role in mouse embryonic stem cell survival and differentiation2017In: Stem Cell Reports, ISSN 2213-6711, Vol. 9, no 1, p. 92-107Article in journal (Refereed)
    Abstract [en]

    THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression.

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  • 19.
    Ahmad, Irfan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Rouf, Syed Fazle
    Sun, Lei
    Cimdins, Annika
    Shafeeq, Sulman
    Le Guyon, Soazig
    Schottkowski, Marco
    Rhen, Mikael
    Romling, Ute
    BcsZ inhibits biofilm phenotypes and promotes virulence by blocking cellulose production in Salmonella enterica serovar Typhimurium2016In: Microbial Cell Factories, E-ISSN 1475-2859, Vol. 15, article id 177Article in journal (Refereed)
    Abstract [en]

    Background: Cellulose, a 1,4 beta-glucan polysaccharide, is produced by a variety of organisms including bacteria. Although the production of cellulose has a high biological, ecological and economical impact, regulatory mechanisms of cellulose biosynthesis are mostly unknown. Family eight cellulases are regularly associated with cellulose biosynthesis operons in bacteria; however, their function is poorly characterized. In this study, we analysed the role of the cellulase BcsZ encoded by the bcsABZC cellulose biosynthesis operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) in biofilm related behavior. We also investigated the involvement of BcsZ in pathogenesis of S. Typhimurium including a murine typhoid fever infection model. Result: In S. Typhimurium, cellulase BcsZ with a putative periplasmic location negatively regulates cellulose biosynthesis. Moreover, as assessed with a non-polar mutant, BcsZ affects cellulose-associated phenotypes such as the rdar biofilm morphotype, cell clumping, biofilm formation, pellicle formation and flagella-dependent motility. Strikingly, although upregulation of cellulose biosynthesis was not observed on agar plate medium at 37 degrees C, BcsZ is required for efficient pathogen-host interaction. Key virulence phenotypes of S. Typhimurium such as invasion of epithelial cells and proliferation in macrophages were positively regulated by BcsZ. Further on, a bcsZ mutant was outcompeted by the wild type in organ colonization in the murine typhoid fever infection model. Selected phenotypes were relieved upon deletion of the cellulose synthase BcsA and/or the central biofilm activator CsgD. Conclusion: Although the protein scaffold has an additional physiological role, our findings indicate that the catalytic activity of BcsZ effectively downregulates CsgD activated cellulose biosynthesis. Repression of cellulose production by BcsZ subsequently enables Salmonella to efficiently colonize the host.

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  • 20.
    Ahrenstedt, Lage
    et al.
    KTH, School of Biotechnology (BIO). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Olksanen, Antti
    VTT Technical Research Centre of Finland.
    Salmien, Kristian
    VTT Technical Research Centre of Finland.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Paper dry strength improvement by xyloglucan addition: Wet-end application, spray coating and synergism with borate2008In: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 62, no 1, p. 8-14Article in journal (Refereed)
    Abstract [en]

    The polysaccharide xyloglucan as a wet-end additive improves paper properties. In the present study, paper strength improvement was analysed for dry handsheets made from chemical, mechanical and recycled pulps coated with xyloglucan in a spray application. Results are compared with sheets made from the same pulps treated with xyloglucan in the wet-end. Kraft pulp handsheets of bleached hardwood and softwood showed significant improvements of tensile, tear and Z-strength by xyloglucan spray treatment versus wet-end application, whereas handsheets of de-inked and thermomechanical pulp were improved only slightly. In both wet-end and spray applications, the effect of xyloglucan addition was intimately related to the presence of non-cellulosic components on the fibre surface. Further strength improvements were obtained for chemical pulps by addition of borax to the spray solution, which were likely to be due to the formation of borate-mediated xyloglucan cross-links. Spray coating of xyloglucan, with or without borax, thus represents a potential new application of this polysaccharide to increase paper dry strength.

  • 21.
    Ain, Noor U.
    et al.
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan; Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
    Muhammad, Niaz
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
    Dianatpour, Mehdi
    Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
    Baroncelli, Marta
    Division of pediatric endocrinology and Center for Molecular Medicine, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden.
    Iqbal, Muddassar
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
    Fard, Mohammad A. F.
    Persian BayanGene Research and Training Center, Shiraz, Iran.
    Bukhari, Ihtisham
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
    Ahmed, Sufian
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
    Hajipour, Massoumeh
    Persian BayanGene Research and Training Center, Shiraz, Iran.
    Tabatabaie, Zahra
    Persian BayanGene Research and Training Center, Shiraz, Iran.
    Foroutan, Hamidreza
    Laparoscopy research center, Shiraz University of Medical Sciences, Shiraz, Iran.
    Nilsson, Ola
    Örebro University, School of Medical Sciences. Division of pediatric endocrinology and Center for Molecular Medicine, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden; Örebro University Hospital, Örebro, Örebro, Sweden.
    Faghihi, Mohammad A.
    Persian BayanGene Research and Training Center, Shiraz, Iran.
    Makitie, Outi
    Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; Folkhälsan Institute of Genetics, Helsinki, Finland.
    Naz, Sadaf
    School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
    Biallelic TMEM251 variants in patients with severe skeletal dysplasia and extreme short stature2020In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 42, no 1, p. 89-101Article in journal (Refereed)
    Abstract [en]

    Skeletal dysplasias are a heterogeneous group of disorders ranging from mild to lethal skeletal defects. We investigated two unrelated families with individuals presenting with a severe skeletal disorder. In family NMD02, affected individuals had a dysostosis multiplex-like skeletal dysplasia and severe short stature (<-8.5 SD). They manifested increasingly coarse facial features, protruding abdomens, and progressive skeletal changes, reminiscent of mucopolysaccharidosis. The patients gradually lost mobility and the two oldest affected individuals died in their twenties. The affected child in family ID01 had coarse facial features and severe skeletal dysplasia with clinical features similar to mucopolysaccharidosis. She had short stature, craniosynostosis, kyphoscoliosis, and hip-joint subluxation. She died at the age of 5 years. Whole-exome sequencing identified two homozygous variants c.133C>T; p.(Arg45Trp) and c.215dupA; p.(Tyr72Ter), respectively, in the two families, affecting an evolutionary conserved gene TMEM251 (NM_001098621.1). Immunofluorescence and confocal studies using human osteosarcoma cells indicated that TMEM251 is localized to the Golgi complex. However, p.Arg45Trp mutant TMEM251 protein was targeted less efficiently and the localization was punctate. Tmem251 knockdown by small interfering RNA induced dedifferentiation of rat primary chondrocytes. Our work implicates TMEM251 in the pathogenesis of a novel disorder and suggests its potential function in chondrocyte differentiation.

  • 22.
    Aisenbrey, Christopher
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Byström, Roberth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oliveberg, Mikael
    Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    SOD1 associates to membranes in its folded apo-stateManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease accompanied by misfolding and intracellular deposition of superoxide dismutase 1 (SOD1). Although the molecular details behind this misfolding process are yet poorly understood, increasing evidence suggest that SOD1 is most susceptible to misfolding in its metal-free and relatively unstable apo-state. Here, we addressed the question, if misfolding and aggregation of SOD1 involves erroneous interactions with membranes as has been implicated for the Aβ peptide in Alzheimers disease. To examine this possibility we subjected various apo SOD1 variants to the presence of different membrane systems. The results reveal that wild type apoSOD1 but to less extent destabilized ALS mutations interact with charged vesicles under physiologically relevant conditions, thereby acquiring pronounced helical structural features. As the data further show, the protein binds to the membranes by an electrostatically driven mechanism, which requires a folded apo-state conformation and a negative membrane surface potential. Unfolded SOD1 molecules show no appreciable affinity to the membrane surfaces yielding a correlation between increased stability, i. e. occupancy of folded molecules and extend of membrane association. Since this trend opposes the correlation between decreased SOD1 stability and progression of neural damage, the results suggest that membrane association is not part of the ALS mechanism. An explanation could be that the observed membrane association of apo SOD1 is reversible and does not ‘bleed out’ in irreversible aggregation as observed for other precursors of protein-misfolding diseases.

  • 23.
    Ajalloueian, F.
    et al.
    Isfahan Univ Technol, Dept Text Engn, Ctr Excellence Appl Nanotechnol, Esfahan, Iran..
    Fransson, M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Tavanai, H.
    Isfahan Univ Technol, Dept Text Engn, Ctr Excellence Appl Nanotechnol, Esfahan, Iran..
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Magnusson, Peetra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala Univ, Dept Immunol Genet & Pathol IGP, Uppsala, Sweden..
    Arpanaei, A.
    Natl Inst Genet Engn & Biotechnol, Dept Ind & Environm Biotechnol, Tehran, Iran..
    Comparing PLGA and PLGA/Chitosan Nanofibers Seeded by Msc: A Cell-scaffold Interaction Study2015In: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 21, p. S406-S407Article in journal (Other academic)
  • 24.
    Ajalloueian, F.
    et al.
    Tech Univ Denmark, Copenhagen, Denmark..
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Fossum, M.
    Karolinska Inst, Stockholm, Sweden..
    Chronakis, I. S.
    Tech Univ Denmark, Copenhagen, Denmark..
    Integrated Micro/Nanofibrous PLGA-Collagen Scaffold: an Optimized Method for Plastic Compression of Collagen into PLGA Microfibers2015In: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 21, p. S347-S347Article in journal (Other academic)
  • 25.
    Akbas, Esvet
    et al.
    Department of Chemistry, Van Yuzuncu Yil University, Van, Türkiye.
    Othman, Khdir A.
    Department of Chemistry, Van Yuzuncu Yil University, Van, Türkiye; Faculty of science and health, Department of chemistry, Koya University, Koy Sanjaq, Iraq.
    Çelikezen, Fatih Çağlar
    Bitlis Eren University Faculty of Science and Letter, Department of Chemistry, Bitlis, Turkey.
    Aydogan Ejder, Nebahat
    Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdoğan University, Rize, Turkey.
    Turkez, Hasan
    Department of Medical Biology, Faculty of Medicine, Atatürk University, Erzurum, Turkey.
    Yapca, Omer Erkan
    Department of Obstetrics and Gynecology, Atatürk University Faculty of Medicine, Erzurum, Turkey.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Centre for Host-Microbiome Interactions Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London, UK.
    Synthesis, Characterization, Theoretical Studies and in Vitro Embriyotoxic, Genotoxic and Anticancer Effects of Novel Phenyl(1,4,6-Triphenyl-2-Thioxo-1,2,3,4-Tetrahydropyrimidin-5-yl)Methanone2023In: Polycyclic aromatic compounds (Print), ISSN 1040-6638, E-ISSN 1563-5333, p. 1-18Article in journal (Refereed)
    Abstract [en]

    In this study, phenyl (1,4,6-triphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone was obtained by using the Biginelli reaction method. The structure of this compound was analyzed using elemental analysis, IR, 1H, and 13C NMR. The quantum chemical calculations (QCC) of this compound were performed density functional theory (DFT) method, 6–31 G (d, p) base set, and B3LYP functions with the Gaussian09W software package. Literature shows that pyrimidine-derived compounds have very active biological properties. For this reason, the biologically active properties of the synthesized compound were also examined. To determine embryotoxic, genotoxic, and cytotoxic effects of compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH) release, micronucleus (MN) and 8-OH-dG assays were carried out. On the other hand, pharmacokinetic and toxicity properties (ADMET) were predicted in silico via SwissADME and Protox-II web tools. In silico estimates of this compound used in the study showed that the compound has the covetable physicochemical properties for bioavailability. In conclusion, the obtained results of our study clearly showed that this compound exerted strong toxicity potential.

  • 26.
    Akhtar, Ahmad Saleem
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Centrifugal microfluidics-based point of care diagnostics at resource limited settings2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Advancements in medical diagnostics have allowed us to understand the underlying mechanism and treat the root cause for many diseases which had been causing morbidity and mortality up until this point in human history. Furthermore, many of the standard diagnostic procedures have now been transformed to provide answers at or near the point-of-care. However, the effects of these positive developments have not trickled down to the parts of our society which are considered underdeveloped and lack the necessary infrastructure and facilities. Diagnostics in such resource limited settings still lag behind and fail to provide the requisite healthcare. 

    In order to translate the diagnostic solutions designed for central laboratories to resource limited settings, there are certain challenges that need to be addressed, such as portability, reduction in cost and ease-of-use, while keeping the sensitivity and specificity at the required level. The work presented in this thesis focuses on addressing some of these issues by using microfluidics to develop diagnostic platforms that are suitable to be used in resource limited settings. 

    In paper I, a very low-cost and simple centrifugal microfluidic platform was developed to be used in settings which do not have a reliable supply of electricity. The platform uses a smartphone as a source of power and the sensors of the phone for speed control.

    In paper II, a portable and low-cost diagnostic platform was developed for multiplexed detection of biomarkers based on centrifugal microfluidics. The platform uses colorimetric detection and a simple readout method which does not require a spectrophotometer for quantification.

    In paper III, a platform was developed for COVID-19 diagnostics which combines centrifugal microfluidics with a novel bead-based strategy for signal enhancement. The platform uses fluorescent detection with a smartphone readout and has the capability to process up to 20 samples at the same time.

    In paper IV, as a follow up of paper III, a more advanced platform was developed for COVID-19 diagnostics which allows the operator to carry out nucleic acid amplification in a completely automated manner, from adding the sample to getting the final result.

    In paper V, an alternative method for detection of SARS-CoV-2 was developed using electrochemical biosensing. This work combines the electrochemical technique with a flexible printed circuit board for a rapid, amplification-free and label-free detection of target SARS-CoV-2 sequences.

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  • 27.
    Akhtar, Ahmad Saleem
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Moura, João Martinho
    International Iberian Nanotechnology Laboratory.
    Paula, Luis
    International Iberian Nanotechnology Laboratory.
    Pedro, Adriano
    International Iberian Nanotechnology Laboratory.
    Martins, Fabio
    International Iberian Nanotechnology Laboratory.
    Mota, Duarte
    International Iberian Nanotechnology Laboratory.
    Pinto, Ines Fernandes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Martins, Marco
    International Iberian Nanotechnology Laboratory.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Fully automated centrifugal microfluidic platform for COVID-19 detection using computer vision-based readoutManuscript (preprint) (Other academic)
    Abstract [en]

    COVID-19 pandemic made it evident that the world is unprepared for effectively tackling a pandemic resulting from an infectious disease. The conventional diagnostic methods for detection of infectious diseases were limited to centralized laboratories. As the burden of testing increased with the spread of the disease, the centralized testing facilities were strained for resources and personnel. These problems were further exacerbated in low- and middle-income countries where the health and transport infrastructure are not very well developed. To overcome this reliance on centralized testing and to facilitate decentralized testing, focus was shifted towards development of novel point-of-care diagnostic methods. We report the development of a fully automated centrifugal microfluidic platform that uses loop mediated isothermal amplification (LAMP) combined with computer vision-based readout for COVID-19 detection. The integrated platform allows sample to answer analysis at the push of a single button and can process 26 samples in 40 minutes. The platform performs a completely automated assay protocol involving heating, rotation and detection without the need for user intervention. A limit of detection of approximately 100 RNA copies in 10 µL reaction was achieved using RNA fragments spiked in water and similar results were obtained for artificial saliva samples. 

  • 28.
    Akhtar, Ahmad Saleem
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Soares, Ruben R. G.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Pinto, Ines Fernandes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Center for the Advancement of Integrated Medical and Engineering Sciences, AIMES.
    A portable and low-cost centrifugal microfluidic platform for multiplexed colorimetric detection of protein biomarkers2023In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1245, article id 340823Article in journal (Refereed)
    Abstract [en]

    Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings.

    Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.

  • 29. Akoachere, Monique
    et al.
    Iozef, Rimma
    Rahlfs, Stefan
    Deponte, Marcel
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Creighton, Donald J
    Schirmer, Heiner
    Becker, Katja
    Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts.2005In: Biol Chem, ISSN 1431-6730, Vol. 386, no 1, p. 41-52Article in journal (Refereed)
    Abstract [en]

    The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.

  • 30.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Boinapally, Vamsi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution2015In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143091Article in journal (Refereed)
    Abstract [en]

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

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  • 31.
    Al-Amin, Abdullah
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gallant, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lööf, Sara
    Department of Oncology-Pathology, Karolinska Institutet.
    Lengqvist, Johan
    Department of Medicine, Karolinska Institutet.
    Bacanu, Smarand
    Department of Oncology-Pathology, Karolinska Institutet.
    Nordlund, Pär
    Department of Oncology-Pathology, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Sensitive Measurement of Cellular Drug-Target Engagement Using Multiplex Proximity Extension AssaysManuscript (preprint) (Other academic)
  • 32.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Gallant, Caroline J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Muthelo, Phathutshedzo M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout2021In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 93, no 31, p. 10999-11009Article in journal (Other academic)
    Abstract [en]

    The ability to monitor target engagement in cellular contexts is a key for successful drug discovery and also valuable in clinical routine. A cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. The CETSA combined with mass spectrometry (MS) detection can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis is slow, requires substantial amounts of the sample material, and often misses proteins of specific interest. Here, we combined the CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of a set of 67 proteins from small amounts of the sample material treated with kinase inhibitors. The results were concordant with the corresponding analyses read out via MS. Our approach allows analyses of large numbers of specific target proteins at high sensitivity in limited sample aliquots. Highly sensitive multiplex CETSA-PEA assays are therefore promising for monitoring drug-target engagement in small sample aliquots in the course of drug development and potentially in clinical settings.

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  • 33.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism. Center for Molecular Medicine, Department of Medicine (Solna), Science for Life Laboratory, Karolinska Institutet.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hammond, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Haybaeck, Johannes
    Institute of Pathology, Neuropathology and Molecular Pathology, Medical University of Innsbruck; Diagnostic and Research Institute of Pathology, Medical University of Graz.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jenmalm Jensen, Annika
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 22, p. e129-e129Article in journal (Refereed)
    Abstract [en]

    Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.

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  • 34.
    Al-Amin, Rasel Abdullah
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Muthelo, Phathutshedzo M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vincke, Cecile
    Structural Biology Research Center, Vrije Universiteit Brussel, Belgium..
    Muyldermans, Serge
    Structural Biology Research Center, Vrije Universiteit Brussel, Belgium.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents2022In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 98, no 28, p. 10054-10061Article in journal (Refereed)
    Abstract [en]

    High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

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  • 35. Al-Anati, Lauy
    et al.
    Viluksela, Matti
    Strid, Anna
    Bergman, Åke
    Andersson, Patrik L
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Stenius, Ulla
    Högberg, Johan
    Hydroxyl metabolite of PCB 180 induces DNA damage signaling and enhances the DNA damaging effect of benzo[a]pyrene2015In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 239, p. 164-173Article in journal (Refereed)
    Abstract [en]

    Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) and their hydroxyl metabolites (OH-PCBs) are ubiquitous environmental contaminants in human tissues and blood. The toxicological impact of these metabolites is poorly understood. In this study rats were exposed to ultrapure PCB180 (10-1000 mg/kg bw) for 28 days and induction of genotoxic stress in liver was investigated. DNA damage signaling proteins (pChk1Ser317 and gamma H2AXSer319) were increased dose dependently in female rats. This increase was paralleled by increasing levels of the metabolite 3'-OH-PCB180. pChk1 was the most sensitive marker. In in vitro studies HepG2 cells were exposed to 1 mu M of PCB180 and 3'-OH-PCB180 or the positive control benzo[a]pyrene (BaP, 5 mu M). 3'-OH-PCB180, but not PCB180, induced CYP1A1 mRNA and gamma H2AX. CYP1A1 mRNA induction was seen at 1 h, and gamma H2AX at 3 h. The anti-oxidant N-Acetyl-L-Cysteine (NAC) completely prevented, and 17 beta-estradiol amplified the gamma H2AX induction by 3'-OH-PCB180. As 3'-OH-PCB180 induced CYP1A1, a major BaP-metabolizing and activating enzyme, interactions between 3'-OH-PCB180 and BaP was also studied. The metabolite amplified the DNA damage signaling response to BaP. In conclusion, metabolism of PCB180 to its hydroxyl metabolite and the subsequent induction of CYP1A1 seem important for DNA damage induced by PCB180 in vivo. Amplification of the response with estradiol may explain why DNA damage was only seen in female rats.

  • 36. Alberro-Brage, Andres
    et al.
    Kryvenko, Vitalii
    Malainou, Christina
    Guenther, Stefan
    Morty, Rory E.
    Seeger, Werner
    Herold, Susanne
    Samakovlis, Christos
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Vadasz, Istvan
    Influenza virus decreases albumin uptake and megalin expression in alveolar epithelial cells2023In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1260973Article in journal (Refereed)
    Abstract [en]

    Introduction

    Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis.

    Methods

    To investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq.

    Results

    IV significantly downregulated albumin uptake, independently of activation of the TGF- β1/GSK3β axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake.

    Discussion

    Our results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.

  • 37.
    Alberti, Esteban
    et al.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Los, Marek Jan
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany; BioApplications Enterprises, Winnipeg, MB, Canada.
    Garcia, Rocio
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Fraga, JL
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Serrano, T.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Hernandez, E.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Macías, R.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Martinez, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Castillo, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    de la Cuétara, K.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
    Prolonged Survival and expression of neural markers by bone marrow-derived stem cells transplanted into brain lesions2009In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 15, no 2, p. BR47-BR54Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease. MATERIAL/METHODS: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR. RESULTS: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA. CONCLUSIONS: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.

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  • 38. Albèr, C.
    et al.
    Brandner, B. D.
    Björklund, S.
    Billsten, P.
    Corkery, Robert
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Engblom, J.
    Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 11, p. 2470-2478Article in journal (Refereed)
    Abstract [en]

    The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.

  • 39. Alcocer, Marcos
    et al.
    Rundqvist, Louise
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ber e 1 protein: the versatile major allergen from Brazil nut seeds.2012In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 34, no 4, p. 597-610Article in journal (Refereed)
    Abstract [en]

    Due mainly to its extremely high content of sulphur amino acids, Ber e 1 protein, the major allergen from Brazil nut, has attracted much scientific and press attention. Ber e 1 was the main target protein in early biotechnology transgenic work, in early processing studies of plant storage proteins, in plant vacuolar targeting studies and as the main protein in early nutritional supplementation experiments. Ber e 1 was also one of the first food allergens to be unintentionally transferred from one plant to another and was involved in the first reported case of systemic allergic reaction caused by a food allergen transferred in semen. In this review, many of the Ber e 1 unique biotechnological and structural functions are discussed with a particular emphasis on its use as model protein for studies of intrinsic allergenicity of food proteins.

  • 40. Alex, Amal
    et al.
    Piano, Valentina
    Polley, Soumitra
    Stuiver, Marchel
    Voss, Stephanie
    Ciossani, Giuseppe
    Overlack, Katharina
    Voss, Beate
    Wohlgemuth, Sabine
    Petrovic, Arsen
    Wu, Yao-Wen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Selenko, Philipp
    Musacchio, Andrea
    Maffini, Stefano
    Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e48287Article in journal (Refereed)
    Abstract [en]

    Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

  • 41.
    Alexander, Helen K.
    et al.
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Booy, Evan P.
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Xiao, Wenyan
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Ezzati, Peyman
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Baust, Heinrich
    Department of Radiooncology, University of Erlangen, Erlangen, Germany .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Selected technologies to control genes and their products for experimental and clinical purposes2007In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 55, no 3, p. 139-149Article in journal (Refereed)
    Abstract [en]

    "On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch (R), coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.

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  • 42.
    Alfredsson-Timmins, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristell, Carolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Henningson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lyckman, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bjerling, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe2009In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 118, no 1, p. 99-112Article in journal (Refereed)
    Abstract [en]

    There are several documented events of changes in subnuclear localization during gene activation. However, there are conflicting data on whether the nuclear periphery is a compartment for gene repression or activation, and whether genes are moved to the pores at the nuclear membrane (NM) or not during gene activation. Nitrogen starvation of fission yeast serves as a good model system for studying gene induction since it causes fast regulation of hundreds of genes. In this study the subnuclear localization of two gene clusters repressed by nitrogen was investigated. During normal growth conditions the gene clusters localized to the nuclear periphery at the opposite side of the nucleus as compared to the spindle pole body (SPB). This constrained localization was dependent on the histone deacetylase Clr3, known to transcriptionally repress genes in these clusters. Already 20 minutes after nitrogen depletion drastic changes in subnuclear localization of the two loci were observed, away from the NM towards the nuclear interior. At least for one of the clusters the movement was clearly transcription dependent. Data presented here illustrates how interconnected events of gene activation and nuclear reorganization are, as well as provides a suggestion of how nuclear organization might be maintained.

  • 43. Ali, Raja Hashim
    et al.
    Muhammad, Sayyed Auwn
    Khan, Mehmood Alam
    Arvestad, Lars
    Stockholm University, Faculty of Science, Numerical Analysis and Computer Science (NADA). Stockholm University, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center, Sweden .
    Quantitative synteny scoring improves homology inference and partitioning of gene families2013In: BMC Bioinformatics, E-ISSN 1471-2105, Vol. 14, no Suppl,15, p. S12-Article in journal (Refereed)
    Abstract [en]

    Background

    Clustering sequences into families has long been an important step in characterization of genes and proteins. There are many algorithms developed for this purpose, most of which are based on either direct similarity between gene pairs or some sort of network structure, where weights on edges of constructed graphs are based on similarity. However, conserved synteny is an important signal that can help distinguish homology and it has not been utilized to its fullest potential.

    Results

    Here, we present GenFamClust, a pipeline that combines the network properties of sequence similarity and synteny to assess homology relationship and merge known homologs into groups of gene families. GenFamClust identifies homologs in a more informed and accurate manner as compared to similarity based approaches. We tested our method against the Neighborhood Correlation method on two diverse datasets consisting of fully sequenced genomes of eukaryotes and synthetic data.

    Conclusions

    The results obtained from both datasets confirm that synteny helps determine homology and GenFamClust improves on Neighborhood Correlation method. The accuracy as well as the definition of synteny scores is the most valuable contribution of GenFamClust.

  • 44.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mitochondrial Peptidasome, PreP, relation to Alzheimer Disease2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid-β (Aβ) is the toxic peptide implicated in the pathogenesis of Alzheimer Disease (AD). Accumulation of Aβ has been shown in brain mitochondria from AD patients and AD mice models. The occurrence of Aβ in the mitochondrial matrix leads to free radical generation and apoptosis in neurons.

    In our studies, Aβ was found in brain mitochondria of living patients with plaque pathology. Extracellular Aβ was taken up by neuroblastoma cells and was found in the mitochondria. Moreover, we showed that Aβ40 and Aβ42 are transported into mitochondria via the Translocase of the Outer Membrane, the TOM machinery.

    We have identified the mitochondrial Aβ-degrading protease hPreP, in human brain mitochondrial matrix. PreP is a metalloprotease, originally identified as presequence protease, but was also shown to degrade other unstructured peptides including Aβ. Immunoinactivation of PreP in human brain mitochondria revealed PreP to be the protease responsible for Aβ degradation in mitochondria.

    Also, we have investigated if genetic variation in the gene encoding hPreP is associated with AD by genotyping 19 single nucleotide polymorphisms (SNPs) in the Swedish population. The study did not show a genetic association between any of the genotyped SNPs and the risk to AD. However, the biochemical analysis of four SNPs selected on the basis of their location within a structural homology model of hPreP revealed a decreased activity compared to wildtype.

    Interestingly, the activity of PreP in human brain mitochondrial matrix in AD individuals was significantly lower compared to non-dement aged-matched controls. These findings were also confirmed in brain mitochondrial matrix of AD mouse models. These results suggest that a decreased PreP activity may contribute to Aβ aggregation and accumulation inside mitochondria leading to neuronal death in AD. In summary, our findings show that the degradation of Aβ by hPreP may be of importance in the pathology of AD.

  • 45.
    Aljabali, Alaa A. A.
    et al.
    Yarmouk Univ, Fac Pharm, Dept Pharmaceut & Pharmaceut Technol, Irbid 21163, Jordan..
    Hassan, Sk Sarif
    Pingla Thana Mahavidyalaya, Dept Math, Paschim Medinipur, India..
    Pabari, Ritesh M.
    Royal Coll Surgeons Ireland, Sch Pharm, Dublin, Ireland..
    Shahcheraghi, Seyed H.
    Shahid Sadoughi Univ Med Sci, Shahid Sadoughi Hosp, Infect Dis Res Ctr, Yazd, Iran..
    Mishra, Vijay
    Lovely Profess Univ, Sch Pharmaceut Sci, Phagwara 144411, Punjab, India..
    Charbe, Nitin B.
    Pontificia Univ Catolica Chile, Fac Quim & Farm, Dept Quim Organ, Santiago 7820436, Chile..
    Chellappan, Dinesh K.
    Int Med Univ, Sch Pharm, Dept Life Sci, Kuala Lumpur 57000, Malaysia..
    Dureja, Harish
    Maharshi Dayanand Univ, Dept Pharmaceut Sci, Rohtak 124001, Haryana, India..
    Gupta, Gaurav
    Suresh Gyan Vihar Univ, Sch Pharm, Mahal Rd, Jaipur 302017, Rajasthan, India..
    Almutary, Abdulmajeed G.
    Qassim Univ, Coll Appl Med Sci, Dept Med Biotechnol, Buraydah, Saudi Arabia..
    Alnuqaydan, Abdullah M.
    Qassim Univ, Coll Appl Med Sci, Dept Med Biotechnol, Buraydah, Saudi Arabia..
    Verma, Suresh K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Panda, Pritam K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Mishra, Yogendra Kumar
    Univ Southern Denmark, Mads Clausen Inst, NanoSYD, Alsion 2, DK-6400 Sonderborg, Denmark..
    Serrano-Aroca, Angel
    Univ Catolica Valencia San Vicente Martir, Ctr Invest Traslac San Alberto Magno, Biomat & Bioengn Lab, Valencia 46001, Spain..
    Dua, Kamal
    Univ Technol, Grad Sch Hlth, Discipline Pharm, Sydney, NSW, Australia..
    Uversky, Vladimir N.
    Univ S Florida, Morsani Coll Med, Dept Mol Med, Tampa, FL 33612 USA..
    Redwan, Elrashdy M.
    King Abdulazizi Univ, Fac Sci, Dept Biol Sci, Jeddah, Saudi Arabia..
    Bahar, Bojlul
    Univ Cent Lancashire, Sch Sport & Hlth Sci, Int Inst Nutr Sci & Food Safety Studies, Preston PR1 2HE, Lancs, England..
    Bhatia, Amit
    Maharaja Ranjit Singh Punjab Tech Univ, Dabwali Rd, Bathinda 151001, Punjab, India..
    Negi, Poonam
    Shoolini Univ Biotechnol & Management Sci, Sch Pharmaceut Sci, Solan 173229, India..
    Goyal, Rohit
    Shoolini Univ Biotechnol & Management Sci, Sch Pharmaceut Sci, Solan 173229, India..
    McCarron, Paul
    Ulster Univ, Sch Pharm & Pharmaceut Sci, Coleraine BT52 1SA, County Londonde, North Ireland..
    Bakshi, Hamid A.
    Ulster Univ, Sch Pharm & Pharmaceut Sci, Coleraine BT52 1SA, County Londonde, North Ireland..
    Tambuwala, Murtaza M.
    Ulster Univ, Sch Pharm & Pharmaceut Sci, Coleraine BT52 1SA, County Londonde, North Ireland..
    The viral capsid as novel nanomaterials for drug delivery2021In: Future Science OA, E-ISSN 2056-5623, Vol. 7, no 9, article id FSO744Article, review/survey (Refereed)
    Abstract [en]

    The purpose of this review is to highlight recent scientific developments and provide an overview of virus self-assembly and viral particle dynamics. Viruses are organized supramolecular structures with distinct yet related features and functions. Plant viruses are extensively used in biotechnology, and virus-like particulate matter is generated by genetic modification. Both provide a material-based means for selective distribution and delivery of drug molecules. Through surface engineering of their capsids, virus-derived nanomaterials facilitate various potential applications for selective drug delivery. Viruses have significant implications in chemotherapy, gene transfer, vaccine production, immunotherapy and molecular imaging. Lay abstract: The purpose of this review is to highlight recent scientific developments and provide an overview of virus self-assembly and viral particle dynamics. Viruses are organized supramolecular structures with distinct yet related features and functions. Plant viruses are extensively used in biotechnology, and virus-like particulate matter is generated by genetic modification. Both provide a material-based means for selective distribution and delivery of drug molecules. Through surface engineering of their capsids, virus-derived nanomaterials facilitate various potential applications for selective drug delivery. Viruses have significant implications in chemotherapy, gene transfer, vaccine production, immunotherapy and molecular imaging. Here we performed a comprehensive database search to review findings in this area, demonstrating that viral nanostructures possess unique properties that make them ideal for applications in diagnostics, cell labeling, contrasting agents and drug delivery structures.

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  • 46.
    Allerbring, E. B.
    et al.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Duru, A. D.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uchtenhagen, H.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Madhurantakam, C.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Tomek, M. B.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Mazumdar, P. A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Friemann, R.
    Univ Gothenburg, Dept Chem Biochem & Biophys, Gothenburg, Sweden..
    Sandalova, T.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uhlin, M.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, Sweden..
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Achour, A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    The unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics and an alternative structural hotspot2011In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 135, p. 70-70Article in journal (Other academic)
  • 47.
    Almamoun, Radwa
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pierozan, Paula
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Environmental Science.
    Karlsson, Oskar
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Environmental Science.
    Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations2024In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738Article in journal (Refereed)
    Abstract [en]

    To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

  • 48.
    Almstedt, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    New targeted therapies for malignant neural tumors: From systematic discovery to zebrafish models2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cancers in the neural system presents a major health challenge. The most aggressive brain tumor in adults, glioblastoma, has a median survival of 15 months and few therapeutic options. High-risk neuroblastoma, a childhood tumor originating in the sympathetic nervous system, has a 5-year survival under 50%, despite extensive therapy. Molecular characterization of these tumors has had some, but so far limited, clinical impact. In neuroblastoma, patients with ALK mutated tumors can benefit from treatment with ALK inhibitors. In glioblastoma, molecular subgroups have not yet revealed any subgroup-specific gene dependencies due to tumor heterogeneity and plasticity. In this thesis, we identify novel treatment candidates for neuroblastoma and glioblastoma. 

    In paper I, we discover novel drug targets for high-risk neuroblastoma by integrating patient data, large-scale pharmacogenomic profiles, and drug-protein interaction maps. Using a novel algorithm, TargetTranslator, we identify more than 80 targets for this patient group. Activation of cannabinoid receptor 2 (CNR2) or inhibition of mitogen-activated protein kinase 8 (MAPK8) reduces tumor growth in zebrafish and mice models of neuroblastoma, establishing TargetTranslator as a useful tool for target discovery in cancer. 

    In paper II, we screen approximately 1500 compounds across 100 molecularly characterized cell lines from patients to uncover heterogeneous responses to drugs in glioblastoma. We identify several connections between pathway activities and drug response. Sensitivity to proteasome inhibition is linked to oxidative stress response and p53 activity in cells, and can be predicted using a gene signature. We also discover sigma receptors as novel drug targets for glioblastoma and find a synergistic vulnerability in targeting cholesterol homeostasis.

    In paper III, we systematically explore novel targets for glioblastoma using an siRNA screen. Downregulation of ZBTB16 decreases cell cycle-related proteins and transcripts in patient-derived glioblastoma cells. Using a zebrafish assay, we find that ZBTB16 promotes glioblastoma invasion in vivo

    In paper IV, we characterized the growth of seven patient-derived glioblastoma cell lines in orthotopic zebrafish xenografts. Using automated longitudinal imaging, we find that tumor engraftment strongly correlates with tumor initiation capacity in mice xenografts and that the heterogeneous response to proteasome inhibitors is maintained in vivo

    In summary, this thesis identifies novel targets for glioblastoma and neuroblastoma using systematic approaches. Treatment candidates are evaluated in novel zebrafish xenograft models that are developed for high-throughput glioblastoma and neuroblastoma drug evaluation. Together, this thesis provides promising evidence of new therapeutic options for malignant neural tumors.

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  • 49.
    Alrifaiy, Ahmed
    et al.
    Umeå University, Faculty of Science and Technology, Centre for Biomedical Engineering and Physics (CMTF).
    Ramser, Kerstin
    Umeå University, Faculty of Science and Technology, Centre for Biomedical Engineering and Physics (CMTF).
    How to integrate a micropipette into a closed microfluidic system: absorption spectra of an optically trapped erythrocyte2011In: Biomedical Optics Express, E-ISSN 2156-7085, Vol. 2, no 8, p. 2299-2306Article in journal (Refereed)
    Abstract [en]

    We present a new concept of integrating a micropipette within a closed microfluidic system equipped with optical tweezers and a UV-Vis spectrometer. A single red blood cell (RBC) was optically trapped and steered in three dimensions towards a micropipette that was integrated in the microfluidic system. Different oxygenation states of the RBC, triggered by altering the oxygen content in the microchannels through a pump system, were optically monitored by a UV-Vis spectrometer. The built setup is aimed to act as a multifunctional system where the biochemical content and the electrophysiological reaction of a single cell can be monitored simultaneously. The system can be used for other applications like single cell sorting, in vitro fertilization or electrophysiological experiments with precise environmental control of the gas-, and chemical content. 

  • 50.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Westerlund, Kristina
    KTH Royal Inst Technol, Div Prot Technol, Sch Biotechnol, Stockholm, Sweden..
    Velletta, Justin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Eriksson Karlström, Amelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Evaluation of affibody molecule-based PNA-mediated radionuclide pretargeting: Development of an optimized conjugation protocol and 177Lu labeling2017In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 54, p. 1-9Article in journal (Refereed)
    Abstract [en]

    Introduction: We have previously developed a pretargeting approach for affibody-mediated cancer therapy based on PNA-PNA hybridization. In this article we have further developed this approach by optimizing the production of the primary agent, Z(HER2.342)-SR-HP1, and labeling the secondary agent, HP2, with the therapeutic radionuclide Lu-177. We also studied the biodistribution profile of Lu-177-HP2 in mice, and evaluated pretargeting with Lu-177-HP2 in vitro and in vivo.

    Methods: The biodistribution profile of Lu-177-HP2 was evaluated in NMRI mice and compared to the previously studied In-111-HP2. Pretargeting using Lu-177-HP2 was studied in vitro using the HER2-expressing cell lines BT-474 and SKOV-3, and in vivo in mice bearing SKOV-3 xenografts.

    Results and conclusion: Using an optimized production protocol for Z(HER2:342)-SR-HP1 the ligation time was reduced from 15 h to 30 min, and the yield increased from 45% to 70%. Lu-177-labeled HP2 binds specifically in vitro to BT474 and SKOV-3 cells pre-treated with Z(HER2:342)-SR-HP1.Lu-177-HP2 was shown to have a more rapid blood clearance compared to In-111-HP2 in NMRI mice, and the measured radioactivity in blood was 0.22 +/- 0.1 and 0.68 +/- 0.07%ID/g for Lu-177- and In-111-HP2, respectively, at 1 h p.i. In contrast, no significant difference in kidney uptake was observed (4.47 +/- 1.17 and 3.94 +/- 0.58%ID/g for Lu-177- and In-111-HP2, respectively, at I h p.i.). Co-injection with either Gelofusine or lysine significantly reduced the kidney uptake for Lu-177-HP2 (1.0 +/- 0.1 and 1.6 +/- 0.2, respectively, vs. 2.97 +/- 0.87%ID/g in controls at 4 h p.i.). Lu-177-HP2 accumulated in SKOV-3 xenografts in BALB/C nu/nu mice when administered after injection of Z(HER2:342)-SR-HP1. Without pre-injection of Z(HER2:342)-SR-HP1, the uptake of Lu-177-HP2 was about 90-fold lower in tumor (0.23 +/- 0.08 vs. 20.7 +/- 3.5%ID/g). The tumor-to-kidney radioactivity accumulation ratio was almost 5-fold higher in the group of mice pre-injected with Z(HER2:342)-SR-HP1. In conclusion, (177)LuHP2 was shown to be a promising secondary agent for affibody-mediated tumor pretargeting in vivo.

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