| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Balan, Mirela, et al.
(författare)
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Noninvasive intravital high-resolution imaging of pancreatic neuroendocrine tumours
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1, s. 14636-14636
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Tidskriftsartikel (refereegranskat)abstract
- Preclinical trials of cancer drugs in animal models are important for drug development. The Rip1Tag2 (RT2) transgenic mouse, a model of pancreatic neuroendocrine tumours (PNET), has provided immense knowledge about PNET biology, although tumour progression occurs in a location inaccessible for real-time monitoring. To overcome this hurdle we have developed a novel platform for intravital 3D imaging of RT2 tumours to facilitate real-time studies of cancer progression. Pre-oncogenic islets retrieved from RT2 mice were implanted into the anterior chamber of the eye (ACE) of host mice, where they engrafted on the iris, recruited blood vessels and showed continuous growth. Noninvasive confocal and two-photon laser-scanning microscopy through the transparent cornea facilitated high-resolution imaging of tumour growth and angiogenesis. RT2 tumours in the ACE expanded up to 8-fold in size and shared hallmarks with tumours developing in situ in the pancreas. Genetically encoded fluorescent reporters enabled high-resolution imaging of stromal cells and tumour cell migration. Sunitinib treatment impaired RT2 tumour angiogenesis and growth, while overexpression of the vascular endothelial growth factor (VEGF)-B increased tumour angiogenesis though tumour growth was impaired. In conclusion, we present a novel platform for intravital high-resolution and 3D imaging of PNET biology and cancer drug assessment.
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| 3. |
- Martowicz, Agnieszka, et al.
(författare)
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Endothelial beta-Catenin Signaling Supports Postnatal Brain and Retinal Angiogenesis by Promoting Sprouting, Tip Cell Formation, and VEGFR (Vascular Endothelial Growth Factor Receptor) 2 Expression
- 2019
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - : LIPPINCOTT WILLIAMS & WILKINS. - 1079-5642 .- 1524-4636. ; 39:11, s. 2273-2288
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Activation of endothelial beta-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/beta-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial beta-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood.Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial beta-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial beta-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial beta-catenin. Importantly, impaired endothelial beta-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium.Conclusions: Our study identifies molecular crosstalk between the Wnt/beta-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial beta-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.
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| 4. |
- Ning, Frank Chenfei
(författare)
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VEGF-B in islet of langerhans : role in vascular signaling and lipid handling
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The discovery of Vascular endothelial growth factor (VEGF)-B’s role in endothelial fatty acid uptake and tissue lipid accumulation opened a new arena for exploration of VEGF-B in lipid metabolism. Indeed, later studies demonstrated the beneficial/therapeutic effects of reducing VEGF-B signaling systemically. However, the tissue specific role of VEGF-B signaling has yet to be determined and furthermore the contribution of tissue specific VEGF-B activity to the systemic effects is also unknown to date. In paper I, we investigated the role of local VEGF-B signaling in pancreatic islets by employing a novel mouse model where Vegfb in pancreatic β-cells were selectively deleted using Cre/loxP technology. We showed that Vegfb is abundantly expressed in both endocrine and exocrine pancreas, but that ablation of Vegfb from β-cells does not affect systemic glucose homeostasis or islet lipid uptake under chow or high-fat diet (HFD) conditions. However, deletion of Vegfb in pancreatic β-cells increased insulin (Ins2) gene expression, which indicates the potential interaction between VEGF-B signaling and transcriptional regulation of insulin, a phenotype warranting further studies. In paper II, we continued to explore the role of pancreatic β-cell derived VEGF-B in systemic homeostasis. In contrast to the mouse model used in paper I, we utilized a mouse model where the human VEGF-B167 isoform is overexpressed in pancreatic β-cells (under the rat insulin promoter). In this study, we could demonstrate that overexpression of VEGF-B in pancreatic β-cells does not affect systemic glucose homeostasis under chow or HFD conditions. Unexpectedly, overexpression of VEGF-B in pancreatic islets led to increased plasma triglycerides. This could indicate VEGF-B’s involvement in plasma lipoprotein synthesis or degradation, and further studies are needed to elucidate this effect. The role of VEGF-B in angiogenesis is somewhat ambiguous. To address this question, in paper III, we deployed a newly developed intravital imaging protocol where isolated mouse islets were transplanted into the anterior chamber of the eye to facilitate the observation of islet vessel development, pharmacological treatment of tumor islets and effects of VEGF-B overexpression in β-cells. We showed that the advantage of this platform comprises high spatial resolution, real-time monitoring of islet development and feasibility for evaluating efficacy of pharmacological treatments of tumor islets. In addition, we observed that overexpression of VEGF-B in tumor islets increased tumor angiogenesis but impaired tumor growth, providing additional information about the role of VEGF-B in pathological angiogenesis. Canonical Wnt/β-catenin signaling is essential for the retinal and cerebral angiogenesis during development. However, little is known regarding the contribution of endothelial β-catenin signaling in postnatal central nervous system (CNS) and potential interaction with other signaling pathways regulating angiogenesis. In paper IV, we studied the role of endothelial β-catenin signaling in central nervous system angiogenesis. We demonstrated that impaired Wnt/β-catenin signaling resulted in reduced postnatal retinal- and cerebral angiogenesis, presumably by diminished VEGFR2 expression. We furthermore identified crosstalk between the Wnt/β-catenin and Notch/VEGFA signaling pathways. In summary, this thesis provides further knowledge about the specific role of VEGF-B signaling in islets of Langerhans, and its effects on systemic glucose homeostasis and lipid handling. Additionally, development of a novel intravital imaging protocol, as well as uncovering of additional roles of endothelial Wnt/β-catenin signaling in CNS, constitute parts of this thesis.
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