| 11. |
- Kelpšas, Vinardas, et al.
(author)
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Evolving escherichia coli host strains for efficient deuterium labeling of recombinant proteins using sodium pyruvate-d3
- 2021
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:18
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Journal article (peer-reviewed)abstract
- Labeling of proteins with deuterium (2H) is often necessary for structural biology tech-niques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typ-ically, expression hosts are grown in a defined medium with heavy water as the solvent, which is supplemented with a deuterated carbon source. Escherichia coli, which is the most widely used host for recombinant protein production, can utilize several compounds as a carbon source. Glycerol-d8 is often used as a carbon source for deuterium labelling due to its lower cost compered to glucose-d7 . In order to expand available options for recombinant protein deuteration, we investigated the possibility of producing a deuterated carbon source in-house. E. coli can utilize pyruvate as a carbon source and pyruvate-d3 can be made by a relatively simple procedure. To circumvent the very poor growth of E. coli in minimal media with pyruvate as sole carbon source, adaptive laboratory evolution for strain improvement was applied. E. coli strains with enhanced growth in minimal pyruvate medium was subjected to whole genome sequencing and the genetic changes were revealed. One of the evolved strains was adapted for the widely used T7 RNA polymerase overexpression systems. Using the improved strain E. coli DAP1(DE3) and in-house produced deuterated carbon source (pyru-vic acid-d4 and sodium pyruvate-d3 ), we produce deuterated (>90%) triose-phosphate isomerase, at quantities sufficient enough for large volume crystal production and subsequent analysis by neutron crystallography.
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| 12. |
- Kimbung, Siker, et al.
(author)
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Assessment of early response biomarkers in relation to long-term survival in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy plus bevacizumab : Results from the Phase II PROMIX trial
- 2018
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In: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 142:3, s. 618-628
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Journal article (peer-reviewed)abstract
- Pathologic complete response (pCR) is a predictor for favorable outcome after neoadjuvant treatment in early breast cancer. Modulation of gene expression may also provide early readouts of biological activity and prognosis, offering the possibility for timely response-guided treatment adjustment. The role of early transcriptional changes in predicting response to neoadjuvant chemotherapy plus bevacizumab was investigated. One-hundred-and-fifty patients with large, operable and locally advanced HER2-negative breast cancer received epirubicin and docetaxel, with the addition of bevacizumab. Patients underwent tumor biopsies at baseline, after Cycle 2 and at the time of surgery. The primary end point, pCR, and its relation with the secondary endpoints event-free survival (EFS), overall survival (OS) and gene expression profiles, are reported. The pCR rate was 13% (95% CI 8.6-20.2), with significantly more pCRs among triple-negative [28% (95% CI 14.8-45.4)] than among hormone receptor positive (HR+) tumors [9% (95% CI 4.6-16.3); (OR=3.9 [CI=1.5-10.3])]. pCR rates were not associated with EFS or OS. PAM50 subtypes significantly changed after Cycle 2 (p=0.03) and an index of absolute changes in PAM50 correlations between these time-points was associated with EFS [HR=0.62 (CI=0.3-1.1)]. In univariable analyses, signatures for angiogenesis, proliferation, estrogen receptor signaling, invasion and metastasis, and immune response, measured after Cycle 2, were associated with pCR in HR+ tumors. Evaluation of changes in molecular subtypes and other signatures early in the course of neoadjuvant treatment may be predictive of pCR and EFS. These factors may help guide further treatment and should be considered when designing neoadjuvant trials.
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| 13. |
- Kirchhoff, Tomas, et al.
(author)
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Breast cancer risk and 6q22.33 : combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2
- 2012
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In: PLOS ONE. - : Public library of science. - 1932-6203. ; 7:6
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Journal article (peer-reviewed)abstract
- Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR) = 1.03, 95% CI 1.00-1.06, p = 0.023). There was evidence for heterogeneity in the ORs among studies (I(2) = 49.3%; p = <0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR = 0.89, 95%CI 0.80-1.00, p = 0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.
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| 14. |
- Nepomuceno, Thales C., et al.
(author)
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BRCA1 frameshift variants leading to extended incorrect protein C termini
- 2023
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In: Human Genetics and Genomics Advances. - : Elsevier. - 2666-2477. ; 4:4
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Journal article (peer-reviewed)abstract
- Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus constitute loss-of-function variants and underscore the need for comprehensive assessment of individual variants.
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| 15. |
- Persdotter, Sofia, et al.
(author)
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Cooperative inhibitory effects of budesonide and formoterol on eosinophil superoxide production stimulated by bronchial epithelial cell conditioned medium
- 2007
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In: International Archives of Allergy and Immunology. - : S. Karger AG. - 1423-0097 .- 1018-2438. ; 143:3, s. 201-210
-
Journal article (peer-reviewed)abstract
- Background: Improved asthma control by combinations of inhaled glucocorticosteroids (GCs) and long-acting beta(2)-agonists (LABAs) includes a reduced frequency and severity of exacerbations. In view of the association of exacerbations with increased airway inflammation, the question has arisen as to whether LABAs are able to complement the known anti- inflammatory activity of GCs. To address this, we studied the effects of a LABA, formoterol (FORM), and a GC, budesonide (BUD), alone and in combination, on bronchial epithelial cell-mediated eosinophil superoxide production in vitro. Methods: We employed 2 experimental approaches. First, superoxide production by human eosinophils incubated with conditioned medium (CM) from human bronchial epithelial cells cultured for 24 h with vehicle, BUD, FORM or BUD + FORM was measured (Epi/Eos assay). Second, eosinophils were stimulated with vehicle-CM to which the drugs were added (Eos assay). Superoxide production was determined as the superoxide dismutase-inhibitable reduction of ferricytochrome C. Results: CM increased eosinophil superoxide generation (p < 0.01) and epithelial-derived granulocyte macrophage colony-stimulating factor was the mediator responsible. In both assays, FORM dose-dependently inhibited eosinophil superoxide similarly and in the same concentration range as BUD. The BUD + FORM combination was more effective than BUD alone, and it completely inhibited CM-induced superoxide production in the Epi/Eos assay, suggesting complementary effects of both drugs on bronchial epithelial cells and eosinophils. Conclusions: The cooperative, inhibitory effects of BUD and FORM on eosinophils and bronchial epithelial cells, in terms of their effects on eosinophil superoxide production, may represent a possible mechanism for the enhanced anti-inflammatory efficacy of BUD and FORM combination therapy of asthma. Copyright (c) 2007 S. Karger AG, Basel
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| 16. |
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| 17. |
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| 18. |
- Vuorela, Mikko, et al.
(author)
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Further evidence for the contribution of the RAD51C gene in hereditary breast and ovarian cancer susceptibility.
- 2011
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In: Breast cancer research and treatment. - : Springer Science and Business Media LLC. - 1573-7217 .- 0167-6806. ; 130:3, s. 1003-1010
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Journal article (peer-reviewed)abstract
- RAD51C, a RAD51 paralogue involved in homologous recombination, is a recently established Fanconi anemia and breast cancer predisposing factor. In the initial report, RAD51C mutations were shown to confer a high risk for both breast and ovarian tumors, but most of the replication studies published so far have failed to identify any additional susceptibility alleles. Here, we report a full mutation screening of the RAD51C gene in 147 Finnish familial breast cancer cases and in 232 unselected ovarian cancer cases originating from Finland and Sweden. In addition, in order to resolve whether common RAD51C SNPs are risk factors for breast cancer, we genotyped five tagging single nucleotide polymorphisms, rs12946522, rs304270, rs304283, rs17222691, and rs28363312, all located within the gene, from 993 Finnish breast cancer cases and 871 controls for cancer associated variants. Whereas, none of the studied common SNPs associated with breast cancer susceptibility, mutation analysis revealed two clearly pathogenic alterations. RAD51C c.-13_14del27 was observed in one familial breast cancer case and c.774delT in one unselected ovarian cancer case, thus confirming that RAD51C mutations are implicated in breast and ovarian cancer predisposition, although their overall frequency seems to be low. Independent identification of the very recently reported RAD51C c.774delT mutation in yet another patient originating from Sweden suggests that it might be a recurrent mutation in that population and should be studied further. The reliable estimation of the clinical implications of carrying a defective RAD51C allele still requires the identification of additional mutation positive families.
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| 19. |
- Wachenfeldt, Anna von
(author)
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Familial breast cancer : risk populations and their surveillance
- 2011
-
Doctoral thesis (other academic/artistic)abstract
- Women carrying mutations in either BRCA 1 or BRCA2 have a lifetime risk of breast cancer of 80%. As little is known about the risk of other malignancies, apart from ovarian/tubal cancer in mutation carriers, the importance of other malignancies in a family with several cases of breast cancer is hard to evaluate. Women at high risk of breast cancer due to family history are offered genetic counselling and surveillance. Whether women looking for oncogenetic counselling are, in terms of socioeconomic status and health-related quality of life, comparable with women in general is not known. Mammography is a widely used screening method to detect breast cancer and has proven to reduce breast cancer mortality in women older than 50 years. The sensitivity of the method is much lower in women with dense breast and in general young women tend to have denser breast than older women. Most women under surveillance in virtue of family history of breast cancer are younger than 50, thus in a group where mammography alone has not been proved to be effective as a single screening method there is a need for other surveillance methods in women at risk of hereditary breast cancer. We identified 803 BRCA 1/2-negative families with two or more cases of breast cancer and at least one additional malignancy. The observed proportion of different non-breast cancer in the study families was compared with the percentage distribution of non-breast cancer tumours in Sweden. Tumours in endometrium were seen in a significantly larger proportion in the study group than in the general population and could not be explained by previously known syndromes or other explanations for being overrepresented. Thus we suggest that endometrial carcinoma and breast cancer constitute a new breast cancer syndrome. In a cross-sectional study aiming to characterize health-related quality of life and socioeconomic status among all healthy women who had ever visited the Oncogenetic Clinic, Department of Oncology, Södersjukhuset in 1998 – 2004, 306 women consented to participate (82.5%). Significantly more women in the study group were cohabiting (74.2 vs. 43.8%), had the highest education level, (56.7 vs. 39.6%) and had the highest household income (36.9 vs. 12.9 %) as compared to the reference population in the same catchment area. Study subjects reported significantly lower levels of health-related quality of life for subscales related to mental health and for general health compared to normative data, but similar levels on subscales related to physical health. Six-hundred-and-thirty-two women (94%) from one counselling clinic consented to participate in a study aiming to find the most sensitive method to detect breast cancer in women with a familiar risk of the disease. Every woman underwent yearly, and blinded to the other methods, mammography, ultrasound and clinical breast exam. This first report describes the study design and the procedure, and the study cohort regarding hereditary pattern and sociodemographics. Further, the associations between breast density, BMI and other breast-cancer risk factors are elucidated. High breast density was associated with low BMI and young age. However, high density was not associated with increasing risk of breast cancer. Ultrasound and clinical breast examination caused substantially more work-up than MG. The number of detected cancers did not differ from the expected numbers. However, it is too early to draw any conclusion about the sensitivity of the three different modalities..
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| 20. |
- Wendt, Camilla, et al.
(author)
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A search for modifying genetic factors in CHEK2:c.1100delC breast cancer patients
- 2021
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11, s. 1-9
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Journal article (peer-reviewed)abstract
- The risk of breast cancer associated with CHEK2:c.1100delC is 2-threefold but higher in carriers with a family history of breast cancer than without, suggesting that other genetic loci in combination with CHEK2:c.1100delC confer an increased risk in a polygenic model. Part of the excess familial risk has been associated with common low-penetrance variants. This study aimed to identify genetic loci that modify CHEK2:c.1100delC-associated breast cancer risk by searching for candidate risk alleles that are overrepresented in CHEK2:c.1100delC carriers with breast cancer compared with controls. We performed whole-exome sequencing in 28 breast cancer cases with germline CHEK2:c.1100delC, 28 familial breast cancer cases and 70 controls. Candidate alleles were selected for validation in larger cohorts. One recessive synonymous variant, rs16897117, was suggested, but no overrepresentation of homozygous CHEK2:c.1100delC carriers was found in the following validation. Furthermore, 11 non-synonymous candidate alleles were suggested for further testing, but no significant difference in allele frequency could be detected in the validation in CHEK2:c.1100delC cases compared with familial breast cancer, sporadic breast cancer and controls. With this method, we found no support for a CHEK2:c.1100delC-specific genetic modifier. Further studies of CHEK2:c.1100delC genetic modifiers are warranted to improve risk assessment in clinical practice.
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