| 1. |
- van den Berg, Susanne, et al.
(författare)
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Improved solubility of TEV protease by directed evolution.
- 2006
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Ingår i: Journal of biotechnology. - Amsterdam : Elsevier BV. - 0168-1656 .- 1873-4863. ; 121:3, s. 291-8
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Tidskriftsartikel (refereegranskat)abstract
- The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.
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| 2. |
- Lundin, Sverker, et al.
(författare)
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Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.
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| 3. |
- Meletis, Konstantinos, et al.
(författare)
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p53 suppresses the self-renewal of adult neural stem cells
- 2006
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 133:2, s. 363-369
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Tidskriftsartikel (refereegranskat)abstract
- There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.
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| 4. |
- Pettersson, Erik, et al.
(författare)
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Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads
- 2008
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 29:2, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.
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| 5. |
- Sahoo, Krushna Chandra, et al.
(författare)
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Dynamics of Household Waste Segregation Behaviour in Urban Community in Ujjain, India : A Framework Analysis
- 2022
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Ingår i: International Journal of Environmental Research and Public Health. - : MDPI. - 1661-7827 .- 1660-4601. ; 19:12
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Tidskriftsartikel (refereegranskat)abstract
- Waste segregation practices must be socially acceptable, affordable, context-specific, and participatory, which is essential for promoting waste segregation. Therefore, this study explored the urban community members’ motivation, opportunity, and household waste segregation ability. We performed a qualitative study in Ujjain city, India. Ten focus group discussions and eight in-depth interviews were conducted with female and male household members in residential and slum areas. All interviews were digitally recorded, transcribed, and translated. We used the thematic framework technique using the Motivation-Opportunity-Ability-Behaviour theory for analysis. Three themes were constructed: motivation, where household members are motivated to sort waste yet fear the consequences of improper sorting; ability, where household waste segregation is rapidly gaining acceptance as a social norm; and opportunities, involving convenient facilities and a social support system for household members towards waste segregation. This study contributes to developing a knowledge base on waste segregation behaviour and a repertoire to facilitate evidence-based management and policymaking. There is a need for educational intervention and women’s self-help groups’ involvement to develop community orientation and waste segregation literacy. Finally, this study emphasizes the importance of all three behavioural change components, i.e., motivation, opportunity, and ability, in managing sustainable waste segregation practices.
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| 6. |
- Sepehri, Sobhan, 1986, et al.
(författare)
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Homogeneous Differential Magnetic Assay
- 2019
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Ingår i: Acs Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 4:9, s. 2381-2388
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Tidskriftsartikel (refereegranskat)abstract
- Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal. Here, we present a homogeneous differential magnetic assay that utilizes a differential magnetic readout that eliminates previous limitations of homogeneous assays. The assay uses a gradiometer sensor configuration combined with precise microfluidic sample handling. This enables simultaneous differential measurement sample containing a synthesized Vibrio cholerae target and a negative control sample, which reduces the background signal and increases the readout speed. Very low concentrations of targets down to femtomolar levels are thus detectable without any additional amplification of the number of targets. Our homogeneous differential magnetic assay method opens new possibilities for rapid and highly sensitive diagnostics at the point of care.
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| 7. |
- Sievertzon, Maria, et al.
(författare)
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Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
- 2005
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 6, s. 55-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.
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| 8. |
- Sievertzon, Maria, et al.
(författare)
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Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
- 2005
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 6:28, s. 13-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.
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| 9. |
- Svensson, Sofie, et al.
(författare)
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Turning food waste to antibacterial and biocompatible fungal chitin/chitosan monofilaments
- 2022
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 209, s. 618-630
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Tidskriftsartikel (refereegranskat)abstract
- Here, cell wall of a zygomycete fungus, Rhizopus delemar, grown on bread waste was wet spun into monofilaments. Using the whole cell wall material omits the common chitosan isolation and purification steps and leads to higher material utilization. The fungal cell wall contained 36.9% and 19.7% chitosan and chitin, respectively. Solid state NMR of the fungal cell wall material confirmed the presence of chitosan, chitin, and other carbohydrates. Hydrogels were prepared by ultrafine grinding of the cell wall, followed by addition of lactic acid to protonate the amino groups of chitosan, and subsequently wet spun into monofilaments. The monofilament inhibited the growth of Bacillus megaterium (Gram+ bacterium) and Escherichia coli (Gram- bacterium) significantly (92.2% and 99.7% respectively). Cytotoxicity was evaluated using an in vitro assay with human dermal fibroblasts, indicating no toxic inducement from exposure of the monofilaments. The antimicrobial and biocompatible fungal monofilaments, open new avenues for sustainable biomedical textiles from abundant food waste.
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| 10. |
- Agerberth, B, et al.
(författare)
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FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.
- 1995
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 92:1, s. 195-9
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Tidskriftsartikel (refereegranskat)abstract
- PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.
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