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- Ansorge, C, et al.
(author)
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Diagnostic value of abdominal drainage in individual risk assessment of pancreatic fistula following pancreaticoduodenectomy
- 2014
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In: The British journal of surgery. - : Oxford University Press (OUP). - 1365-2168 .- 0007-1323. ; 101:2, s. 100-108
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Journal article (peer-reviewed)abstract
- BackgroundThe use of prophylactic abdominal drainage following pancreaticoduodenectomy (PD) is controversial as its therapeutic value is uncertain. However, the diagnosis of postoperative pancreatic fistula (POPF), the main cause of PD-associated morbidity, is often based on drain pancreatic amylase (DPA) levels. The aim of this study was to assess the predictive value of DPA, plasma pancreatic amylase (PPA) and serum C-reactive protein (CRP) for diagnosing POPF after PD.MethodsPatients undergoing PD with prophylactic drainage between 2008 and 2012 were studied prospectively. DPA, PPA and CRP levels were obtained daily. Differences between groups with clinically relevant POPF (International Study Group on Pancreatic Fistula (ISGPF) grade B/C) and without clinically relevant POPF (no POPF or ISGPF grade A) were evaluated. Receiver operating characteristic (ROC) analyses were performed to determine the value of DPA, PPA and CRP in prediction of POPF. Risk profiles for clinically relevant POPF were constructed and related to the intraoperative pancreatic risk assessment.ResultsFifty-nine (18·7 per cent) of 315 patients developed clinically relevant POPF. DPA, PPA and CRP levels on postoperative day (POD) 1–3 differed significantly between the study groups. In predicting POPF, the DPA level on POD 1 (cut-off at 1322 units/l; odds ratio (OR) 24·61, 95 per cent confidence interval 11·55 to 52·42) and POD 2 (cut-off at 314 units/l; OR 35·45, 14·07 to 89·33) was superior to that of PPA on POD 1 (cut-off at 177 units/l; OR 13·67, 6·46 to 28·94) and POD 2 (cut-off at 98 units/l; OR 16·97, 8·33 to 34·59). When DPA was combined with CRP (cut-off on POD 3 at 202 mg/l; OR 16·98, 8·43 to 34·21), 90·3 per cent of postoperative courses could be predicted correctly (OR 44·14, 16·89 to 115·38).ConclusionThe combination of serum CRP and DPA adequately predicted the development of clinically relevant pancreatic fistula following PD.
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| 2. |
- Aoki, Y, et al.
(author)
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Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation
- 2021
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In: Frontiers in physiology. - : Frontiers Media SA. - 1664-042X. ; 12, s. 689179-
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Journal article (peer-reviewed)abstract
- Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.
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| 6. |
- Corso, G, et al.
(author)
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Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography
- 2017
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 11561-
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.
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| 10. |
- Gustafsson, O, et al.
(author)
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Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP
- 2021
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In: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:6
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Journal article (peer-reviewed)abstract
- The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.
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