Cervical cancer detection by DNA methylation analysis in urine.

Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (r = 0.508-0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744-0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.

Selective Functionalization with PNA of Silicon Nanowires on Silicon Oxide Substrates.

Silicon nanowire chips can function as sensors for cancer DNA detection, whereby selective functionalization of the Si sensing areas over the surrounding silicon oxide would prevent loss of analyte and thus increase the sensitivity. The thermal hydrosilylation of unsaturated carbon-carbon bonds onto H-terminated Si has been studied here to selectively functionalize the Si nanowires with a monolayer of 1,8-nonadiyne. The silicon oxide areas, however, appeared to be functionalized as well. The selectivity toward the Si-H regions was increased by introducing an extra HF treatment after the 1,8-nonadiyne monolayer formation. This step (partly) removed the monolayer from the silicon oxide regions, whereas the Si-C bonds at the Si areas remained intact. The alkyne headgroups of immobilized 1,8-nonadiyne were functionalized with PNA probes by coupling azido-PNA and thiol-PNA by click chemistry and thiol-yne chemistry, respectively. Although both functionalization routes were successful, hybridization could only be detected on the samples with thiol-PNA. No fluorescence was observed when introducing dye-labeled noncomplementary DNA, which indicates specific DNA hybridization. These results open up the possibilities for creating Si nanowire-based DNA sensors with improved selectivity and sensitivity.

Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.

Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.

Assembly and Mechanical Properties of the Cargo-Free and Cargo-Loaded Bacterial Nanocompartment Encapsulin.

Prokaryotes mostly lack membranous compartments that are typical of eukaryotic cells, but instead, they have various protein-based organelles. These include bacterial microcompartments like the carboxysome and the virus-like nanocompartment encapsulin. Encapsulins have an adaptable mechanism for enzyme packaging, which makes it an attractive platform to carry a foreign protein cargo. Here we investigate the assembly pathways and mechanical properties of the cargo-free and cargo-loaded nanocompartments, using a combination of native mass spectrometry, atomic force microscopy and multiscale computational molecular modeling. We show that encapsulin dimers assemble into rigid single-enzyme bacterial containers. Moreover, we demonstrate that cargo encapsulation has a mechanical impact on the shell. The structural similarity of encapsulins to virus capsids is reflected in their mechanical properties. With these robust mechanical properties encapsulins provide a suitable platform for the development of nanotechnological applications.

Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens.

Self-sorting of foreign proteins in a bacterial nanocompartment.

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis.

Predicting the loading of virus-like particles with fluorescent proteins.

The virus-like particle (VLP) of the Cowpea Chlorotic Mottle Virus (CCMV) has often been used to encapsulate foreign cargo. Here we show two different rational design approaches, covalent and noncovalent, for loading teal fluorescent proteins (TFP) into the VLP. The covalent loading approach allows us to gain control over capsid loading on a molecular level. The achieved loading control is used to accurately predict the loading of cargo into CCMV VLP. The effects of molecular confinement were compared for the differently loaded VLPs created with the covalent method. We see that the loading of more than 10 fluorescent proteins in the 18 nm internal cavity of the CCMV capsid gives rise to a maximum efficiency of homo-FRET between the loaded proteins, as measured by fluorescence anisotropy. This shows that already at low levels of VLP loading molecular crowding starts to play a role.