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Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Three-dimensional (3D) cell culture conditions are often used to promote the differentiation of human cells as a prerequisite for the study of organotypic functions and environment-specific cellular responses. Here, we assessed the molecular and functional phenotype of vascular smooth muscle cells (VSMCs) cultured as 3D multilayered aggregates. Microarray studies revealed that these conditions decrease the expression of genes associated with cell cycle control and DNA replication and cease proliferation of VSMCs. This was accompanied by a lower activity level of the mitogen-activated protein kinase ERK1/2 and an increase in autocrine TGFβ/SMAD2/3-mediated signaling - a determinant of VSMC differentiation. However, inhibition of TGFβ signaling did not affect markers of VSMC differentiation such as smooth muscle myosin heavy chain (MYH11) but stimulated pro-inflammatory NFκB-associated gene expression in the first place while decreasing the protein level of NFKB1/p105 and NFKB2/p100 - inhibitors of NFκB transcriptional activity. Moreover, loss of TGFβ signaling also revived VSMC proliferation in 3D aggregates. In conclusion, assembly of VSMCs in multilayered aggregates alters their transcriptome to translate the cellular organization into a resting phenotype. In this context, TGFβ signaling appears to attenuate cell growth and NFκB-controlled gene expression representing important aspects of VSMC quiescence., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The validation of microRNAs (miRNAs) identified by next generation sequencing involves amplification-free and hybridization-based detection of transcripts as criteria for confirming valid miRNAs. Since respective validation is frequently not performed, miRNA repositories likely still contain a substantial fraction of false positive candidates while true miRNAs are not stored in the repositories yet. Especially if downstream analyses are performed with these candidates (e.g. target or pathway prediction), the results may be misleading. In the present study, we evaluated 558 mature miRNAs from miRBase and 1,709 miRNA candidates from next generation sequencing experiments by amplification-free hybridization and investigated their distributions in patients with various disease conditions. Notably, the most significant miRNAs in diseases are often not contained in the miRBase. However, these candidates are evolutionary highly conserved. From the expression patterns, target gene and pathway analyses and evolutionary conservation analyses, we were able to shed light on the complexity of miRNAs in humans. Our data also highlight that a more thorough validation of miRNAs identified by next generation sequencing is required. The results are available in miRCarta ( https://mircarta.cs.uni-saarland.de ).

Breast cancer is a heterogeneous disease with distinct molecular subtypes including the aggressive subtype triple-negative breast cancer (TNBC). We compared blood-borne miRNA signatures of early-stage basal-like (cytokeratin-CK5-positive) TNBC patients to age-matched controls. The miRNAs of TNBC patients were assessed prior to and following platinum-based neoadjuvant chemotherapy (NCT). After an exploratory genome-wide study on 21 cases and 21 controls using microarrays, the identified signatures were verified independently in two laboratories on the same and a new cohort by RT-qPCR. We differentiated the blood of TNBC patients before NCT from controls with 84% sensitivity. The most significant miRNA for this diagnostic classification was miR-126-5p (two tailed t-test p-value of 1.4 × 10 -5 ). Validation confirmed the microarray results for all tested miRNAs. Comparing cancer patients prior to and post NCT highlighted 321 significant miRNAs (among them miR-34a, p-value of 1.2 × 10 -23 ). Our results also suggest that changes in miRNA expression during NCT may have predictive potential to predict pathological complete response (pCR). In conclusion we report that miRNA expression measured from blood facilitates early and minimally-invasive diagnosis of basal-like TNBC. We also demonstrate that NCT has a significant influence on miRNA expression. Finally, we show that blood-borne miRNA profiles monitored over time have potential to predict pCR.

Background: Different work flows have been proposed to use miRNAs as blood-borne biomarkers. In particular, the method used for collecting blood from patients can considerably influence the diagnostic results., Methods: We explored whether dried blood spots (DBSs) facilitate stable miRNA measurements and compared its technical stability with biological variability. First, we tested the stability of DBS samples by generating from 1 person 18 whole-genome-wide miRNA profiles of DBS samples that were exposed to different temperature and humidity conditions. Second, we investigated technical reproducibility by performing 7 replicates of DBS again from 1 person. Third, we investigated DBS samples from 53 patients with lung cancer undergoing different therapies. Across these 3 stages, 108 genome-wide miRNA profiles from DBS were generated and evaluated biostatistically., Results: In the stability analysis, we observed that temperature and humidity had an overall limited influence on the miRNomes (average correlation between the different conditions of 0.993). Usage of a silica gel slightly diminished DBS' technical reproducibility. The 7 technical replicates had an average correlation of 0.996. The correlation with whole-blood PAXGene miRNomes of the same individual was remarkable (correlation of 0.88). Finally, evaluation of the samples from the 53 patients with lung cancer exposed to different therapies showed that the biological variations exceeded the technical variability significantly ( P < 0.0001), yielding 51 dysregulated miRNAs., Conclusions: We present a stable work flow for profiling of whole miRNomes on the basis of samples collected from DBS. Biological variations exceeded technical variations significantly. DBS-based miRNA profiles will potentially further the translational character of miRNA biomarker studies., (© 2017 American Association for Clinical Chemistry.)

Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10-16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).

Background: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists., Methods: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR., Results: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 (95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait., Conclusions: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers.

Objectives: The Stenting of Saphenous Grafts-Xience V (SOS-Xience V) trial prospectively examined the frequency of angiographic in-stent restenosis in saphenous vein graft (SVG) lesions 12 months after implantation of a Xience V everolimus-eluting stent (EES; Abbott Vascular). Optical coherence tomography (OCT) during follow-up angiography was added to the protocol after OCT was approved for clinical use in the United States., Methods: Forty patients with 40 SVG lesions were enrolled in the study, of whom 27 underwent 12-month coronary angiography and 12 (only 1 of whom had in-stent restenosis) also had follow-up OCT evaluation. OCT strut-level analysis was performed to determine the percentage of strut coverage, malapposition, strut protrusion, neointimal thickness, and the existence of thrombus., Results: Mean patient age was 67 ± 7 years, and 95% were men. A total of 2584 struts were evaluated by OCT. The percentages for uncovered, malapposed, and protruding struts were 4%, 9%, and 15%, respectively. The mean strut neointimal thickness was 0.094 ± 0.094 mm. Of the 12 stents analyzed, 4 (33%) showed full neointimal coverage, 2 (17%) had all the struts embedded, 7 (58%) had at least 1 malapposed strut, and 10 (83%) had at least 1 protruding strut. The mean difference between the stent area and the lumen area was 0.36 ± 1.6 mm². No thrombus was detected in the stented areas., Conclusions: Use of EES in SVGs is associated with high rates of stent strut coverage and high malapposition rates at 12 months post implantation.

Working with wavelengths shorter than the deep ultraviolet involves the development of dedicated optics for free-electron lasers with devoted coating techniques and characterizations. High-performance deep-ultraviolet optics are specially developed to create low-loss, high-reflectivity dielectric mirrors with long lifetimes in harsh synchrotron radiation environments. In February 2001, lasing at 189.7 nm, the shortest wavelength obtained so far with free-electron-laser oscillators, was obtained at the European Free-electron-laser project at ELETTRA Synchrotron Light Laboratory, Trieste, Italy. In July 2001, 330-mW extracted power at 250 nm was measured with optimized transmission mirrors. Research and development of coatings correlated to lasing performance are reported.

The authors report eight instances of ECT-induced postictal (emergence) delirium that occurred after bilateral ECT, right unilateral ECT, or left unilateral ECT. They conclude that postictal delirium is a random phenomenon unrelated to lateralized hemispheric mechanisms.