CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

Retrospective evaluation of established cut-off values for the sFlt-1/PlGF ratio for predicting imminent delivery in preeclampsia patients.

OBJECTIVE: The outcome of preeclampsia is difficult to predict. Laboratory markers such as soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PlGF) are thought to be predictive factors. A 2012 study by Verlohren et al. evaluated cut-off values for assessing time to delivery in patients with elevated sFlt-1/PlGF ratios. The present study aimed to evaluate findings in patients with elevated sFlt-1/PlGF ratios who gave birth at Kepler University Hospital in Linz, Austria. The hypothesis was tested, that our patients show longer pregnancy duration despite elevated sFlt-1/PlGF ratios. STUDY DESIGN: This retrospective data analysis included all patients with sFlt-1/PlGF ratios above the established cut-off values between January 2014 and October 2017. Two groups were analyzed relative to gestational age and were matched with healthy controls: 24 + 0 to 33 + 6 gestational weeks, sFlt-1/PlGF ratio >655.2; and 34 + 0 to 36 + 6 gestational weeks, sFlt-1/PlGF ratio >201. MAIN OUTCOME MEASURES: sFlt-1/PlGF ratio and time to delivery correlation. RESULTS: In the <34-week group, 43.2% of the patients delivered beyond 48 h, with a mean sFlt-1/PlGF ratio of 885.06, showing a significantly lower sFlt-1/PlGF ratio than patients who delivered within 2 days (P = 0.04). In the >34-week group, 66.7% were still pregnant after 48 h, with a mean sFlt-1/PlGF ratio of 273.7. CONCLUSION: The sFlt-1/PlGF ratio appears to be a powerful tool for diagnosing and predicting preeclampsia. However, the data do not confirm the cut-off values described earlier, with longer pregnancy durations in this group of patients.

Oncoplastic breast surgery versus conventional breast-conserving surgery: a comparative retrospective study.

BACKGROUND: In addition to conventional breast-conserving surgery (BCS), oncoplastic breast surgery (OBS) is an operation technique that strives simultaneously to increase oncological safety and patient's satisfaction. It is the combination of the best-proven techniques in plastic surgery with surgery for breast cancer. In a growing number of indications, OBS overcomes the limit of conventional BCS by allowing larger resection volumes while avoiding deformities. The aim of our retrospective study (2012-2014) was to compare oncological outcomes of OBS versus BCS. METHODS: We compared two groups of patients with primary non-metastatic breast tumours: group A (n = 291), where BCS was performed, versus group B (n = 52), where OBS was performed. Surgical interventions were performed in German and Swiss teaching hospital settings. The surgeon for group B had subspecialist training in OBS. We assessed outcome in term of re-excision rates, resection margin and complications. RESULTS: Groups were homogenous (no significant differences in terms of age, tumour size, tumour type or grade). The resection margin was larger in group B (7 mm) than in group A (3 mm). Re-excision rate of group B (8%) was significantly lower than in group A (31%). Complication rates were comparably low in groups A and B. CONCLUSION: Despite the limits of retrospective design, our study confirms that OBS is safe and reduces the re-excision rates and the need for further surgery. OBS has the potential to improve oncological care and should be more widely adopted.

The in vivo performance of magnetic particle-loaded injectable, in situ gelling, carriers for the delivery of local hyperthermia.

We investigated the use of in situ implant formation that incorporates superparamagnetic iron oxide nanoparticles (SPIONs) as a form of minimally invasive treatment of cancer lesions by magnetically induced local hyperthermia. We developed injectable formulations that form gels entrapping magnetic particles into a tumor. We used SPIONs embedded in silica microparticles to favor syringeability and incorporated the highest proportion possible to allow large heating capacities. Hydrogel, single-solvent organogel and cosolvent (low-toxicity hydrophilic solvent) organogel formulations were injected into human cancer tumors xenografted in mice. The thermoreversible hydrogels (poloxamer, chitosan), which accommodated 20% w/v of the magnetic microparticles, proved to be inadequate. Alginate hydrogels, however, incorporated 10% w/v of the magnetic microparticles, and the external gelation led to strong implants localizing to the tumor periphery, whereas internal gelation failed in situ. The organogel formulations, which consisted of precipitating polymers dissolved in single organic solvents, displayed various microstructures. A 8% poly(ethylene-vinyl alcohol) in DMSO containing 40% w/v of magnetic microparticles formed the most suitable implants in terms of tumor casting and heat delivery. Importantly, it is of great clinical interest to develop cosolvent formulations with up to 20% w/v of magnetic microparticles that show reduced toxicity and centered tumor implantation.

Local moderate magnetically induced hyperthermia using an implant formed in situ in a mouse tumor model.

PURPOSE: We investigate a new heat delivery technique for the local treatment of solid tumors. The technique involves injecting a formulation that solidifies to form an implant in situ. This implant entraps superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microbeads for magnetically induced moderate hyperthermia. Particle entrapment prevents phagocytosis and distant migration of SPIONs. The implant can be repeatedly heated by magnetic induction. METHODS: We evaluated heating and treatment efficacies by means of thermometry and survival studies in nude mice carrying subcutaneous human colocarcinomas. At day 1, we injected the formulation into the tumor. At day 2, a single 20-min hyperthermia treatment was delivered by 141-kHz magnetic induction using field strengths of 9 to 12 mT under thermometry. RESULTS: SPIONs embedded in silica microbeads were effectively confined within the implant at the injection site. Heat-induced necro-apoptosis was assessed by histology on day 3. On average, 12 mT resulted in tumor temperature of 47.8 degrees C, and over 70% tumor necrosis that correlated to the heat dose (AUC = 282 degrees C.min). In contrast, a 9-mT field strength induced tumoral temperature of 40 degrees C (AUC = 131 degrees C.min) without morphologically identifiable necrosis. Survival after treatment with 10.5 or 12 mT fields was significantly improved compared to non-implanted and implanted controls. Median survival times were 27 and 37 days versus 12 and 21 days respectively. CONCLUSION: Five of eleven mice (45%) of the 12 mT group survived one year without any tumor recurrence, holding promise for tumor therapy using magnetically induced moderate hyperthermia through injectable implants.

Surface triggering receptor expressed on myeloid cells 1 expression patterns in septic shock.

OBJECTIVE: To analyze the pattern of cell-surface expression of the triggering receptor expressed on myeloid cells (TREM) 1 during septic shock. DESIGN AND SETTING: Prospective clinical study in an adult 16-bed medical ICU. PATIENTS AND METHODS: 25 septic shock patients, 15 patients with shock of noninfectious origin and 7 healthy volunteers. Arterial blood was drawn within 12 h of admission and subjected to flow cytometry analysis after staining with anti-TREM-1 and anti-CD14 antibodies. Repeated sampling was performed on days 2, 3, 5, 7, and 14 in septic shock patients. RESULTS: Monocytic TREM-1 expression was significantly higher in septic shock patients (mean fluorescence intensity 2.3+/-0.2) than in nonseptic patients (1.0+/-0.1), and healthy volunteers (1.0+/-0.1). There was no difference in monocytic TREM-1 expression between nonseptic patients and healthy volunteers or between any of the three groups with respect to TREM-1 expression on neutrophils. The time course of TREM-1 expression on monocytes diverged significantly by day 3 between survivors and ns. CONCLUSIONS: The specificity of TREM-1 regulation by infection is highlighted. Moreover, surface TREM-1 expression on monocytes may prove useful in allowing the follow-up of septic patients during the course of the disease.