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| 2. |
- Bergquist, Helen, 1979-
(författare)
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Development of benzoquinoquinoxaline derivatives as triplex-specific probes : Recognition of DNA structures at repeats sequences
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Repeat sequences are associated with several human diseases, such as Friedreich’s ataxia, polycystic kidney disease and cancer. These sequences can form non-B-DNA structures, including triplex (H-DNA) DNA, and are associated with genomic instability and altered gene expression. The occurrence of triplex structures in vivo and identification of their links to biological processes have been challenging. The lack of effective probes has restrained the study of triplex structures in living cells. Here, the triplex binding small molecule benzoquinoquinoxaline (BQQ) and its derivatives were developed as tools to study triplex formation at genomic repeat sequences. The triplex binding efficiency towards both purine and pyrimidine triplex motifs was determined for BQQ, the DNA cleaving BQQ-1,10-(ortho)-phenanthroline (BQQ-OP) and the fluorescent BQQ-Bodipy compounds. BQQ was shown to have the most stabilising effect on both triplex motifs. Moreover, H-DNA structure formation at a pkd1 derived sequence was demonstrated for the first time by BQQ-OP at physiologically relevant conditions. H-DNA formation was also shown at (GAA)n repeats associated with Friedreich’s ataxia and the structure was further analysed on one nucleotide resolution, confirming that (GAA)n repeats form a pyrimidine H-DNA. However, a mixture of different isomers formed at longer (GAA)n repeats. To this end, the interaction between the peptide nucleic acids (PNA) and BQQ was investigated. PNA is a DNA mimic that binds sequence-specifically to dsDNA and can form several PNA-DNA complexes. The results of PNA binding to frataxin (GAA)n expansion in plasmid were evaluated, and in the presence of GAA-PNA no triplex structure could be detected by BQQ-OP cleavage. When the structure formed in the presence of either GAA-PNA or CTT-PNA was further analysed, it was found that GAA-PNA formed a duplex invasion complex preventing H-DNA formation, whereas CTT-PNA formed a triplex invasion complex.
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| 3. |
- Bergquist, Helen, et al.
(författare)
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Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)(n) Repeats by PNA or LNA Targeting
- 2016
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Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Expansion of (GAA)(n) repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)(n) expansions form non-canonical structures, including intramolecular triplex (H-DNA), and Rloops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)(n) expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)(n) repeats of different lengths (short: n= 9, medium: n= 75 or long: n= 115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA) 4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT) 5-PNA did not. We present evidence that (GAA) 4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT) 5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.
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| 4. |
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| 6. |
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| 7. |
- Bergquist, Helen, et al.
(författare)
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Structural Insights into Human Adenovirus Type 4 Virus-Associated RNA I
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:6
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Tidskriftsartikel (refereegranskat)abstract
- RNA molecules can adopt specific RNA triplex structures to execute critical biological functions. Human adenoviruses (HAdVs) are abundant pathogens encoding the essential, noncoding virus-associated RNA I (VA RNAI). Here, we employ a triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinoxaline 1, 10-phenanthroline (BQQ-OP), to unravel a potential RNA triplex formation in VA RNAI. The BQQ-OP cleavage of the pathogenic HAdV type 4 (HAdV-4) VA RNAI indicates that a potential triplex is formed involving the highly conserved stem 4 of the central domain and side stem 7. Further, the integrity of the HAdV-4 VA RNAI side stem 7 contributes to a potential triplex formation in vitro and virus growth in vivo. Collectively, we propose that the HAdV-4 VA RNAI can potentially form a biologically relevant triplex structure.
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| 8. |
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| 9. |
- Bergquist, Helen, 1979-, et al.
(författare)
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Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives
- 2009
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 10:16, s. 2629-2637
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Tidskriftsartikel (refereegranskat)abstract
- Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)nrepeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)nbinding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)nsequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)nrepeats under physiologically relevant conditions.
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| 10. |
- Deng, Tingzhi, et al.
(författare)
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Hippocampal Transcriptome-Wide Association Study Reveals Correlations Between Impaired Glutamatergic Synapse Pathway and Age-Related Hearing Loss in BXD-Recombinant Inbred Mice
- 2021
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Ingår i: Frontiers in Neuroscience. - : Frontiers Media S.A.. - 1662-4548 .- 1662-453X. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Age-related hearing loss (ARHL) is associated with cognitive dysfunction; however, the detailed underlying mechanisms remain unclear. The aim of this study is to investigate the potential underlying mechanism with a system genetics approach. A transcriptome-wide association study was performed on aged (12-32 months old) BXD mice strains. The hippocampus gene expression was obtained from 56 BXD strains, and the hearing acuity was assessed from 54 BXD strains. Further correlation analysis identified a total of 1,435 hearing-related genes in the hippocampus (p < 0.05). Pathway analysis of these genes indicated that the impaired glutamatergic synapse pathway is involved in ARHL (p = 0.0038). Further gene co-expression analysis showed that the expression level of glutamine synthetase (Gls), which is significantly correlated with ARHL (n = 26, r = -0.46, p = 0.0193), is a crucial regulator in glutamatergic synapse pathway and associated with learning and memory behavior. In this study, we present the first systematic evaluation of hippocampus gene expression pattern associated with ARHL, learning, and memory behavior. Our results provide novel potential molecular mechanisms involved in ARHL and cognitive dysfunction association.
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| 11. |
- Eeg-Olofsson, Måns, 1967, et al.
(författare)
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TTCOV19: timing of tracheotomy in SARS-CoV-2-infected patients: a multicentre, single-blinded, randomized, controlled trial
- 2022
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Ingår i: Critical Care. - : Springer Science and Business Media LLC. - 1364-8535. ; 26:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Critically ill COVID-19 patients may develop acute respiratory distress syndrome and the need for respiratory support, including mechanical ventilation in the intensive care unit. Previous observational studies have suggested early tracheotomy to be advantageous. The aim of this parallel, multicentre, single-blinded, randomized controlled trial was to evaluate the optimal timing of tracheotomy. Methods: SARS-CoV-2-infected patients within the Region Vastra Gotaland of Sweden who needed intubation and mechanical respiratory support were included and randomly assigned to early tracheotomy (<= 7 days after intubation) or late tracheotomy (>= 10 days after intubation). The primary objective was to compare the total number of mechanical ventilation days between the groups. Results: One hundred fifty patients (mean age 65 years, 79% males) were included. Seventy-two patients were assigned to early tracheotomy, and 78 were assigned to late tracheotomy. One hundred two patients (68%) underwent tracheotomy of whom sixty-one underwent tracheotomy according to the protocol. The overall median number of days in mechanical ventilation was 18 (IQR 9; 28), but no significant difference was found between the two treatment regimens in the intention-to-treat analysis (between-group difference:- 1.5 days (95% CI -5.7 to 2.8); p= 0.5). A significantly reduced number of mechanical ventilation days was found in the early tracheotomy group during the per-protocol analysis (between-group difference: - 8.0 days (95% CI - 13.8 to - 2.27); p= 0.0064). The overall correlation between the timing of tracheotomy and days of mechanical ventilation was significant (Spearman's correlation: 0.39, p < 0.0001). The total death rate during intensive care was 32.7%, but no significant differences were found between the groups regarding survival, complications or adverse events. Conclusions: The potential superiority of early tracheotomy when compared to late tracheotomy in critically ill patients with COVID-19 was not confirmed by the present randomized controlled trial but is a strategy that should be considered in selected cases where the need for MV for more than 14 days cannot be ruled out.
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| 12. |
- Ghidini, Alice, et al.
(författare)
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Clamping of RNA with PNA enables targeting of microRNA
- 2016
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 14:23, s. 5210-5213
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Tidskriftsartikel (refereegranskat)abstract
- To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.
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| 13. |
- Larsson, Helen, 1982, et al.
(författare)
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Dysphagia and health-related of life in patients with eosinophilic esophagitis: a long-term follow-up.
- 2015
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Ingår i: European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery. - : Springer Science and Business Media LLC. - 1434-4726. ; 272:12, s. 3833-3839
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Tidskriftsartikel (refereegranskat)abstract
- Eosinophilic esophagitis (EoE) is a chronic immune/antigen-mediated disease, with dysphagia as the main symptom. The aim of this study was to survey symptoms and health-related quality of life in adult patients with EoE at least 1year after diagnosis and a 2-month course of topical corticosteroids. Forty-seven consecutive patients [79% males, mean age 49years (range 18-90years)] were evaluated using three different questionnaires at three different occasions: the Watson Dysphagia Scale (WDS), the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Oesophageal Module 18 (EORTC QLQ-OES18) and the Short Form-36 (SF-36). The median time from diagnosis to the long-term follow-up was 23months (range 12-34months). The WDS scores and the EORTC QLQ-OES18 Dysphagia and Eating scale scores were improved after 2months of treatment (p=0.00007, p=0.01, p=0.004, respectively), as were the long-term follow-up scores (p=0.01, p=0.03, p=0.005, respectively), relative to the scores at diagnosis. In addition, the EORTC QLQ-OES18 Choking scores were improved after the steroid course (p=0.003) but not after the long-term follow-up. No significant differences were detected with respect to the SF-36 scores. In summary, EoE seems to be associated with a substantial burden of symptoms that improve significantly after treatment. A partial remission persists more than 1year after diagnosis and the discontinuation of medication. The WDS and the EORTC QLQ-OES18 appear to be sensitive instruments appropriate for surveillance in these patients.
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| 14. |
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| 15. |
- Larsson, Helen, 1982, et al.
(författare)
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Grade of eosinophilia versus symptoms in patients with dysphagia and esophageal eosinophilia.
- 2016
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Ingår i: Diseases of the esophagus. - : Oxford University Press (OUP). - 1120-8694. ; 29:8, s. 971-976
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to assess whether the symptom severity and health-related quality of life (HRQL) of patients with dysphagia and esophageal eosinophilia correlate with disease activity as expressed by the number of eosinophils in the esophageal mucosa. This study included newly diagnosed (n = 58) or relapsed patients (n = 7), where 40% were diagnosed in connection with esophageal bolus impaction. The mean age was 45 years (19-88), and 74% were men. Symptoms and HRQL were recorded using the Watson Dysphagia Scale (WDS), the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire - Oesophageal Module 18 and the Short Form-36 Questionnaire. Histological samples gathered from the proximal and distal esophageal mucosa were stained using both hematoxylin and eosin (HE) and an immunohistochemical (IHC) technique against 'Eosinophil Major Basic Protein,' and the peak number of eosinophils per high-power field was assessed. More eosinophils were detected after IHC staining than HE staining (P < 0.001). No correlation was found between symptoms or the HRQL and the number of eosinophils. However, higher numbers of eosinophils at the proximal esophagus were found in patients with concomitant bolus impaction (IHC P < 0.05 and HE P < 0.05) and could serve as a risk marker.
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| 16. |
- Liu, Jian, et al.
(författare)
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Quantitative proteomics approach reveals novel biomarkers and pathological mechanism of keloid
- 2022
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Ingår i: PROTEOMICS - Clinical Applications. - : John Wiley & Sons. - 1862-8346 .- 1862-8354. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Background Keloid is a pathological skin scar formation with complex and unclear molecular pathology mechanism. Novel biomarkers and associated mechanisms are needed to improve current therapies.Objectives To identify novel biomarkers and underlying pathological mechanisms of keloids.MethodsSix pairs of keloid scar tissues and corresponding normal skin tissues were quantitatively analyzed by a high-resolution label-free mass spectrometry-based proteomics approach. Differential protein expression data was further analyzed by a comprehensive bioinformatics approach to identify novel biomarkers and mechanistic pathways for keloid formation. Candidate biomarkers were validated experimentally.Results In total, 1359 proteins were identified by proteomic analysis. Of these, 206 proteins exhibited a significant difference in expression between keloid scar and normal skin tissues. RCN3 and CALU were significantly upregulated in keloids. RCN1 and PDGFRL were uniquely expressed in keloids. Pathway analysis suggested that the XBP1-mediated unfolded protein response (UPR) pathway was involved in keloid formation. Moreover, a PDGFRL centric gene coexpression network was constructed to illustrate its function in skin.Conclusions and Clinical Relevance Our study proposed four novel biomarkers and highlighted the role of XBP1-mediated UPR pathway in the pathology of keloids. It provided novel biological insights that contribute to develop novel therapeutic strategies for keloids.
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| 17. |
- Plate, John, 1993, et al.
(författare)
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Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3′-diaminobenzidine-stained esophageal biopsies
- 2024
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Ingår i: DISEASES OF THE ESOPHAGUS. - 1120-8694 .- 1442-2050.
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Tidskriftsartikel (refereegranskat)abstract
- Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) share many histopathological features; therefore, markers for differentiation are of diagnostic interest and may add to the understanding of the underlying mechanisms. The nitrergic system is upregulated in GERD and probably also in EoE. Esophageal biopsies of patients with EoE (n = 20), GERD (n = 20), and healthy volunteers (HVs) (n = 15) were exposed to antibodies against inducible nitric oxide synthase (iNOS), nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3. The stained object glasses were randomized, digitized, and blindly analyzed regarding the expression of DAB (3,3 '-diaminobenzidine) by a protocol developed in QuPath software. A statistically significant overexpression of iNOS was observed in patients with any of the two inflammatory diseases compared with that in HVs. Eotaxin-3 could differentiate HVs versus inflammatory states. Gastroesophageal reflux patients displayed the highest levels of nitrotyrosine. Neither iNOS nor nitrotyrosine alone were able to differentiate between the two diseases. For that purpose, eosinophil peroxidase was a better candidate, as the mean levels increased stepwise from HVs via GERD to EoE. iNOS and nitrotyrosine are significantly overexpressed in patients with EoE and GERD compared with healthy controls, but only eosinophil peroxidase could differentiate the two types of esophagitis. The implications of the finding of the highest levels of nitrotyrosine among gastroesophageal reflux patients are discussed.
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| 18. |
- Terenius, Olle, et al.
(författare)
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Midgut bacterial dynamics in aedes aegypti
- 2012
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Ingår i: FEMS Microbiology Ecology. - : Oxford University Press (OUP). - 0168-6496 .- 1574-6941. ; 80:3, s. 556-565
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Tidskriftsartikel (refereegranskat)abstract
- In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on LuriaBertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.similar to aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.similar to aegypti survive better in Ae.similar to aegypti as compared to P.similar to stewartii isolated from the malaria mosquito Anopheles gambiae.
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| 19. |
- Umek, Tea, et al.
(författare)
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Oligonucleotide Binding to Non-B-DNA in MYC
- 2019
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 24:5
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Tidskriftsartikel (refereegranskat)abstract
- MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major MYC promoters P-1 and P-2. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.
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