| 1. |
- Alalam, Hanna, et al.
(författare)
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A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance.
- 2020
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Ingår i: mSystems. - 2379-5077. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes' effects in liquid mating assays and complement one of the novel genes' effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.
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| 2. |
- Bäckberg, Matilda, et al.
(författare)
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Using in vitro receptor activity studies of synthetic cannabinoids to support the risk assessment of new psychoactive substances – A Swedish strategy to protect public health from harm
- 2023
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Ingår i: Forensic Science International. - : Elsevier Ireland Ltd. - 0379-0738 .- 1872-6283. ; 348
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Tidskriftsartikel (refereegranskat)abstract
- In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited. © 2023 The Authors
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| 3. |
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| 4. |
- Kmoch, Laura, 1987, et al.
(författare)
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Access mapping highlights risks from land reform in upland Myanmar
- 2021
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Ingår i: Journal of Land Use Science. - 1747-4248 .- 1747-423X. ; 16:1, s. 34-54
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Tidskriftsartikel (refereegranskat)abstract
- Secure land access is vital for Myanmar's upland households, who rely on crops and forests to meet their subsistence needs. But recent land reforms threaten to undermine customary tenure and land-use practices in Myanmar. This paper combines income accounting methods with access theory to assess how new legislation may affect four Chin communities in the country's north-west. Our assessment of 94 households' land-access mechanisms and economic benefits from different types of land reveals existing land-access inequalities among Chin households and demonstrates communities' continued dependence on environmental resources, especially those from swidden fields, home gardens and forests. A majority of households would lose all of their land-derived income, if they were denied access to communities' customarily governed land, e.g., under the Vacant, Fallow and Virgin Land Management Law. Policy stakeholders should therefore intervene, to alleviate land-access inequalities among Chin households and to direct Myanmar's land-system dynamics onto more just development trajectories.
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| 5. |
- Kmoch, Laura, 1987, et al.
(författare)
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Cyclone Komen’s aftermath: Local knowledge shows how poverty and inequalities fuel climate risk in western Myanmar
- 2021
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Ingår i: Regional Environmental Change. - : Springer Science and Business Media LLC. - 1436-378X .- 1436-3798. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- Cyclones and other extreme events exert increasing pressure on South-East Asia’s societies and put smallholder farmers at risk. Here, we draw on participatory causal-diagramming workshops, interviews and survey data, to provide contextually grounded knowledge about rural communities’ exposure and vulnerability to climate-related hazards in western Myanmar. By tracing how the 2015 cyclone Komen led to a prolonged humanitarian disaster, we show that climate-related risks in this area arise from the complex interplay of households’ pre-existing vulnerabilities, persistent farming challenges, extensive disasters and cascading effects, which disparately affect lowland and upland communities. The different household strata’s dissimilar vulnerabilities vis-à-vis Komen’s impacts were rooted in the distinct exposure of their production systems to landslides and floods. Pre-existing land-access barriers, land-degradation processes, climatic stressors, agricultural pests and diseases, and chronic lack of assets and food insecurity further mediated households’ vulnerability. Relief interventions did not stop the disaster’s escalation, although this could have been achieved with early technical and material assistance to address the cyclone’s impacts on farmers’ land. Targeted aid for households facing imminent food insecurity or debt crisis could have lessened engagement in precarious coping strategies and distress migration. A diversification of households’ livelihood and land-use practices and increased redundancies of critical assets and infrastructure could help to mitigate future cyclone-triggered disasters. By demonstrating the strengths of local knowledge approaches in untangling the complex interplay of extreme events with households’ everyday vulnerabilities and agricultural land-use practices, we make a case for more contextually grounded disaster risk and climate adaptation research.
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| 6. |
- Nilsson, Anna Matilda, et al.
(författare)
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Mixed Airway and Pulmonary Parenchymal Disease in Patients With Primary Sjögren Syndrome : A 6-year Follow-up
- 2021
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Ingår i: Journal of Rheumatology. - Toronto, Canada : Journal Of Rheumatology Publishing Co. - 0315-162X .- 1499-2752. ; 48:2, s. 232-240
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Tidskriftsartikel (refereegranskat)abstract
- To assess pulmonary function and chronic obstructive pulmonary disease (COPD) development over time in patients with primary Sjögren syndrome (pSS), as well as the association between pulmonary function, radiographic findings, respiratory symptoms, and clinical features of pSS, taking cigarette consumption into account.
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| 7. |
- Sahi, Maryam, et al.
(författare)
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Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy
- 2024
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2024:203
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) ˂14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.
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| 8. |
- Thomsen, Hanna, 1989, et al.
(författare)
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Increased antibiotic efficacy and noninvasive monitoring of Staphylococcus epidermidis biofilms using per-cysteamine-substituted γ-cyclodextrin - A delivery effect validated by fluorescence microscopy.
- 2020
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Ingår i: International journal of pharmaceutics. - : Elsevier BV. - 1873-3476 .- 0378-5173. ; 587
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Tidskriftsartikel (refereegranskat)abstract
- Limited and poor delivery of antibiotics is cited as one reason for the difficulty in treating antibiotic-resistant biofilms associated with chronic infections. We investigate the effectiveness of a positively charged, single isomer cyclodextrin derivative, octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) to improve the delivery of antibiotics to biofilms. Using multiphoton laser scanning microscopy complemented with super-resolution fluorescence microscopy, we showed that γCys tagged with fluorescein (FITC) is uniformly distributed throughout live S. epidermidis biofilm cultures in vitro and results suggest it is localized extracellularly in the biofilm matrix. NMR spectroscopic data in aqueous solution confirm that γCys forms inclusion complexes with both the antibiotics oxacillin and rifampicin. Efficacy of γCys/antibiotic (oxacillin and rifampicin) was measured in the biofilms. While treatment with γCys/oxacillin had little improvement over oxacillin alone, γCys/rifampicin reduced the biofilm viability to background levels demonstrating a remarkable improvement over rifampicin alone. The strong synergistic effect for γCys/rifampicin is at this stage not clearly understood, but plausible explanations are related to increased solubility of rifampicin upon complexation and/or synergistic interference with components of the biofilm. The results demonstrate that designed cyclodextrin nanocarriers, like γCys, efficiently deliver suitable antibiotics to biofilms and that fluorescence microscopy offers a novel approach for mechanistic investigations.
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| 9. |
- Tijani, Muyideen Kolapo, et al.
(författare)
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How to Detect Antibodies Against Babesia divergens in Human Blood Samples
- 2024
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Ingår i: Open Forum Infectious Diseases. - : OXFORD UNIV PRESS INC. - 2328-8957. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Background. Today only indirect fluorescent antibody assays (IFAs) are commercially available to detect antibodies against Babesia divergens in humans. IFA is subjective and requires highly experienced staff. We have therefore developed an enzyme-linked immunosorbent assay (ELISA)-based method for measuring anti-B. divergens immunoglobulin G antibodies in human blood samples. Methods. Crude merozoite extract from in vitro cultures of a new B. divergens isolate was used in ELISA to detect antibodies in different sets of samples: Borrelia burgdorferi-positive samples, healthy individuals, tick-bitten individuals including follow-up samples 3 months later, positive control samples from patients with an active Babesia infection, and samples from malaria-endemic regions. As a reference, IFA was used to detect antibodies in the tick-bitten samples. Western blot was used to evaluate reactions against specific bands in extracts with/without parasites. Results. Using IFA as the reference method, the sensitivity and specificity of the ELISA were 86% (12/14) and 100% (52/52). There was a very high correlation (r = -0.84; P = .0004) between IFA dilution factors and ELISA absorbances among the samples classified as positive. Five percent of the B. burgdorferi-positive samples were judged as weakly positive and 5% as strongly positive in our ELISA. Western blot showed that the immunodominant antigens (∼120 kDa) were from merozoites and not from erythrocytes. Conclusions. This ELISA can detect antibodies directed against B. divergens, and it can be a useful and easy assay to handle compared with IFA. The ELISA can also measure high and low levels of antibodies, which could give insight into the recency of a B. divergens infection.
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