| 1. |
- Niklasson, Mia, et al.
(författare)
-
Mesenchymal transition and increased therapy resistance of glioblastoma cells is related to astrocyte reactivity
- 2019
-
Ingår i: Journal of Pathology. - : WILEY. - 0022-3417 .- 1096-9896. ; 249:3, s. 295-307
-
Tidskriftsartikel (refereegranskat)abstract
- Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1 beta). IL1 beta induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called 'reactive GICs'. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. (c) 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
|
|
| 2. |
- Segerman, Anna, et al.
(författare)
-
Clonal Variation in Drug and Radiation Response among Glioma-Initiating Cells Is Linked to Proneural-Mesenchymal Transition
- 2016
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 17:11, s. 2994-3009
-
Tidskriftsartikel (refereegranskat)abstract
- Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuumof multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.
|
|
| 3. |
|
|
| 4. |
- Ekman, Lisa, et al.
(författare)
-
A shotgun metagenomic investigation of the microbiota of udder cleft dermatitis in comparison to healthy skin in dairy cows
- 2020
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15
-
Tidskriftsartikel (refereegranskat)abstract
- Udder cleft dermatitis (UCD) is a skin condition affecting the fore udder attachment of dairy cows. UCD may be defined as mild (eczematous skin changes) or severe (open wounds, large skin changes). Our aims were to compare the microbiota of mild and severe UCD lesions with the microbiota of healthy skin from the fore udder attachment of control cows, and to investigate whether mastitis-causing pathogens are present in UCD lesions. Samples were obtained from cows in six dairy herds. In total, 36 UCD samples categorized as mild (n = 17) or severe (n = 19) and 13 control samples were sequenced using a shotgun metagenomic approach and the reads were taxonomically classified based on their k-mer content. The Wilcoxon rank sum test was used to compare the abundance of different taxa between different sample types, as well as to compare the bacterial diversity between samples. A high proportion of bacteria was seen in all samples. Control samples had a higher proportion of archaeal reads, whereas most samples had low proportions of fungi, protozoa and viruses. The bacterial microbiota differed between controls and mild and severe UCD samples in both composition and diversity. Subgroups of UCD samples were visible, characterized by increased proportion of one or a few bacterial genera or species, e.g. Corynebacterium, Staphylococcus, Brevibacterium luteolum, Trueperella pyogenes and Fusobacterium necrophorum. Bifidobacterium spp. were more common in controls compared to UCD samples. The bacterial diversity was higher in controls compared to UCD samples. Bacteria commonly associated with mastitis were uncommon. In conclusion, a dysbiosis of the microbiota of mild and severe UCD samples was seen, characterized by decreased diversity and an increased proportion of certain bacteria. There was no evidence of a specific pathogen causing UCD or that UCD lesions are important reservoirs for mastitis-causing bacteria.
|
|
| 5. |
- Geisler-Lee, Jane, et al.
(författare)
-
Poplar carbohydrate-active enzymes. Gene identification and expression analyses.
- 2006
-
Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 140:3, s. 946-62
-
Tidskriftsartikel (refereegranskat)abstract
- Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr. & Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar (Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis (Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.
|
|
| 6. |
- Holmfeldt, Per, et al.
(författare)
-
The Schistosoma mansoni protein SM16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity
- 2007
-
Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 156:2, s. 225-234
-
Tidskriftsartikel (refereegranskat)abstract
- Sm16/SmSLP/SPO-1 (Sm16) has been identified as a developmentally regulated protein that is released from specific glands of the Schistosoma mansoni parasite during skin penetration. Sm16 has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. Here we used a cell line to confirm signal peptide-dependent secretion and to define the secreted form of Sm16 for production in E. coli. We present evidence from both in vitro experiments and studies on transfected human cells that refute any functional similarity with stathmin/Op18. Instead of an Op18-like activity, we found that targeting of Sm16 to the cytosol of human cells, which was achieved by ectopic expression of Sm16 lacking the signal peptide, results in a caspase-dependent apoptotic response. Interestingly, by analysis of recombinant preparations we found that the secreted form of Sm16 is a lipid bilayer-binding protein that efficiently binds to the surface of diverse cell types by a polyanion-independent mechanism, which results in uptake by endocytosis. While the significance of the pro-apoptotic activity exerted by cytosolic Sm16 remains unclear, the present findings on cell-surface-binding properties of Sm16 seems likely to be of functional relevance during skin penetration of the parasite.
|
|
| 7. |
- Kamel, Wael, et al.
(författare)
-
Small RNA Sequence Analysis of Adenovirus VA RNA-Derived MiRNAs Reveals an Unexpected Serotype-Specific Difference in Structure and Abundance
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:8, s. e105746-
-
Tidskriftsartikel (refereegranskat)abstract
- Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs). We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3'-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5'-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3'-mivaRNAs with a slight variation of the position of the 5'-terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5'-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.
|
|
| 8. |
- Kamel, Wael, et al.
(författare)
-
The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells
- 2013
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:9, s. 4802-4812
-
Tidskriftsartikel (refereegranskat)abstract
- At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.
|
|
| 9. |
- Kotova, Irina, et al.
(författare)
-
A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
- 2001
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 268:16, s. 4527-4536
-
Tidskriftsartikel (refereegranskat)abstract
- Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.
|
|
| 10. |
- Segerman, Bo, et al.
(författare)
-
Characterization of genes with tissue-specific differential expression patterns in Populus
- 2007
-
Ingår i: Tree Genetics & Genomes. - : Springer Science and Business Media LLC. - 1614-2942 .- 1614-2950. ; 3, s. 351-362
-
Tidskriftsartikel (refereegranskat)abstract
- Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns.
|
|
| 11. |
- Segerman, Bo, et al.
(författare)
-
The efficiency of Nextera XT tagmentation depends on G and C bases in the binding motif leading to uneven coverage in bacterial species with low and neutral GC-content
- 2022
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Whole-genome sequencing (WGS) is becoming the new standard for bacterial high-resolution typing and the performance of laboratories is being evaluated in interlaboratory comparisons. The use of the Illumina Nextera XT library preparation kit has been found to be associated with poorer performance due to a GC-content-dependent coverage bias. The bias is especially strong when sequencing low GC-content species. Here, we have made an in-depth analysis of the Nextera XT coverage bias problem using data from a proficiency test of the low GC-content species Campylobacter jejuni. We have compared Nextera XT with Nextera Flex/DNA Prep and examined the consequences on downstream WGS analysis when using different quantities of raw data. We have also analyzed how the coverage bias relates to differential usage of tagmentation cleavage sites. We found that the tagmentation site was characterized by a symmetrical motif with a central AT-rich region surrounded by Gs and Cs. The Gs and Cs appeared to be the main determinant for cleavage efficiency and the genomic regions that were associated with low coverage only contained low-efficiency cleavage sites. This explains why low GC-content genomes and regions are more subjected to coverage bias. We furthermore extended our analysis to other datasets representing other bacterial species. We visualized how the coverage bias was large in low GC-content species such as C. jejuni, C. coli, Staphylococcus aureus, and Listeria monocytogenes, whereas species with neutral GC-content such as Salmonella enterica and Escherichia coli were only affected in certain regions. Species with high GC-content such as Mycobacterium tuberculosis and Pseudomonas aeruginosa were hardly affected at all. The coverage bias associated with Nextera XT was not found when Nextera Flex/DNA Prep had been used.
|
|
| 12. |
- Segerman, Bo
(författare)
-
The Most Frequently Used Sequencing Technologies and Assembly Methods in Different Time Segments of the Bacterial Surveillance and RefSeq Genome Databases
- 2020
-
Ingår i: Frontiers in Cellular and Infection Microbiology. - : FRONTIERS MEDIA SA. - 2235-2988. ; 10
-
Forskningsöversikt (refereegranskat)abstract
- Whole genome sequencing has become a powerful tool in modern microbiology. Especially bacterial genomes are sequenced in high numbers. Whole genome sequencing is not only used in research projects, but also in surveillance projects and outbreak investigations. Many whole genome analysis workflows begins with the production of a genome assembly. To accomplish this, a number of different sequencing technologies and assembly methods are available. Here, a summarization is provided over the most frequently used sequence technology and genome assembly approaches reported for the bacterial RefSeq genomes and for the bacterial genomes submitted as belonging to a surveillance project. The data is presented both in total and broken up on a per year basis. Information associated with over 400,000 publically available genomes dated April 2020 and prior were used. The information summarized include (i) the most frequently used sequencing technologies, (ii) the most common combinations of sequencing technologies, (iii) the most reported sequencing depth, and (iv) the most frequently used assembly software solutions. In all, this mini review provides an overview of the currently most common workflows for producing bacterial whole genome sequence assemblies.
|
|
| 13. |
- Tuskan, G A, et al.
(författare)
-
The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).
- 2006
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 313:5793, s. 1596-604
-
Tidskriftsartikel (refereegranskat)abstract
- We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.
|
|
| 14. |
- Westergren Jakobsson, Amanda, et al.
(författare)
-
The Human Adenovirus 2 Transcriptome : An Amazing Complexity of Alternatively Spliced mRNAs
- 2021
-
Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 95:4
-
Tidskriftsartikel (refereegranskat)abstract
- We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.
|
|
| 15. |
- Xu, Ning, et al.
(författare)
-
Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the RNA-induced silencing complex and associate with polyribosomes
- 2007
-
Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 81:19, s. 10540-10549
-
Tidskriftsartikel (refereegranskat)abstract
- Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.
|
|
