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Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

The demand for strong, compact permanent magnets essential for the energy transition drives innovation in magnet manufacturing. Additive manufacturing, particularly Powder Bed Fusion of metals using a laser beam (PBF‐LB/M), offers potential for near‐net‐shaped Nd‐Fe‐B permanent magnets but often falls short compared to conventional methods. A less explored strategy to enhance these magnets is feedstock modification with nanoparticles. It is demonstrated that modifying a Nd‐Fe‐B‐based feedstock with 1 wt.% Ag nanoparticles boost the coercivity of the magnets to a record value of 935 ± 6 kA m−1 without further post‐processing or heat treatments. Suitable volumetric energy densities for the PBF‐LB/M process are determined using finite element simulations predicting melt pool behavior and part density. Microstructural analyses reveal finer grain sizes and more equiaxed nanocrystalline structures due to the modification. Atom probe tomography identifies three phases in the Ag‐modified samples, with Ag forming nanophase regions with rare‐earth elements near the amorphous Zr‐Ti‐B‐rich intergranular phase, potentially decoupling the Nd2Fe14B primary phase. The study shows that superior magnetic properties primarily result from microstructure modification rather than part density. These findings highlight inventive material design approaches via feedstock surface modification to achieve superior magnetic performance in additively manufactured Nd‐Fe‐B magnets. Innovative manufacturing using Laser Powder Bed Fusion (PBF‐LB/M) and 1 wt.% Ag nanoparticle modification boosts Nd‐Fe‐B magnets' coercivity to a record value of 935 ± 6 kA m−1, achieved without post‐processing or heat treatments. Microstructural analyses (SEM‐EBSD, HR‐TEM, atom probe tomography) reveal fine, equiaxed grains driving this enhancement, showcasing a very promising novel feedstock surface modification strategy.

Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Tungsten-modified hydrogenated amorphous carbon coatings (a-C:H:W) were deposited on high speed steel by reactive magnetron sputtering of a tungsten carbide target in an argon-ethine atmosphere. The deposition parameters, sputtering power, bias voltage, argon and ethine flow rate, were varied according to a central composite design comprising 25 different parameter combinations. For comparison, a tungsten carbide coating was deposited, as well. During coating deposition, the process variables, total pressure, sputtering voltage and bias current, were measured as process characteristics. The thickness of the deposited coatings was determined using the crater grinding method, and the deposition rate was calculated. Young’s modulus E and indentation hardness HIT were characterized by means of nanoindentation. With E = 80