| 1. |
- Andersson, Sandra, et al.
(författare)
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Insufficient antibody validation challenges oestrogen receptor beta research
- 2017
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
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| 2. |
- Cheng, Yirui, et al.
(författare)
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Comparison of serum exosome isolation methods on co-precipitated free microRNAs.
- 2020
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Ingår i: PeerJ. - : PeerJ. - 2167-8359. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.Methods: In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods.Results: Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.
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| 3. |
- Hakim Jaffer Ali, Mohammed, et al.
(författare)
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Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater
- 2021
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Ingår i: Science of the Total Environment. - : Elsevier BV. - 0048-9697 .- 1879-1026. ; 755
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Tidskriftsartikel (refereegranskat)abstract
- Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.
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| 4. |
- Odeberg, J, et al.
(författare)
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Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene : implications for mutation detection.
- 1999
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 235:1-2
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Tidskriftsartikel (refereegranskat)abstract
- We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.
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| 5. |
- Richter, Karin, et al.
(författare)
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Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
- 2006
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 7, s. 75-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.
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| 6. |
- Vivekanand, Aashlesha Chekkala, et al.
(författare)
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Statistical Analysis of SARS-CoV-2 Using Wastewater-Based Data of Stockholm, Sweden
- 2023
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Ingår i: International Journal of Environmental Research and Public Health. - : MDPI AG. - 1661-7827 .- 1660-4601. ; 20:5
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Tidskriftsartikel (refereegranskat)abstract
- An approach based on wastewater epidemiology can be used to monitor the COVID-19 pandemic by assessing the gene copy number of SARS-CoV-2 in wastewater. In the present study, we statistically analyzed such data from six inlets of three wastewater treatment plants, covering six regions of Stockholm, Sweden, collected over an approximate year period (week 16 of 2020 to week 22 of 2021). SARS-CoV-2 gene copy number and population-based biomarker PMMoV, as well as clinical data, such as the number of positive cases, intensive care unit numbers, and deaths, were analyzed statistically using correlations and principal component analysis (PCA). Despite the population differences, the PCA for the Stockholm dataset showed that the case numbers are well grouped across wastewater treatment plants. Furthermore, when considering the data from the whole of Stockholm, the wastewater characteristics (flow rate m3/day, PMMoV Ct value, and SARS-CoV gene copy number) were significantly correlated with the public health agency’s report of SARS-CoV-2 infection rates (0.419 to 0.95, p-value < 0.01). However, while the PCA results showed that the case numbers for each wastewater treatment plant were well grouped concerning PC1 (37.3%) and PC2 (19.67%), the results from the correlation analysis for the individual wastewater treatment plants showed varied trends. SARS-CoV-2 fluctuations can be accurately predicted through statistical analyses of wastewater-based epidemiology, as demonstrated in this study.
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| 7. |
- Williams, Cecilia, 1969-, et al.
(författare)
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A high frequency of sequence alterations is due to formalin fixation of archival specimens.
- 1999
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Ingår i: American Journal of Pathology. - 0002-9440 .- 1525-2191. ; 155:5
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Tidskriftsartikel (refereegranskat)abstract
- Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.
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