ancer immunotherapies have shown promise in harnessing the immune system to target and destroy cancers, leading to clinical benefit enriched in patients with a high mutational burden [1][2][3][4][5] . Multiple studies indicate that cytotoxic CD8 T cells targeting tumor neoantigens are critical to tumor control and clearance in response to immunotherapies targeting CTLA-4 or PD-1 [6][7][8][9][10] . Clinical responses to CPI therapy rely mostly on reinvigorating preexisting tumor-specific T cell responses 11 , and active vaccination to expand preexisting and prime de novo tumor-specific T cells is anticipated to overcome this limitation.The limited success of cancer vaccines in the past can be attributed to a number of factors, including selection of poorly immunogenic self-antigens 12 , insufficiently immunogenic vaccine platforms and immunosuppressive milieus in patients with advanced cancers 4 . Accordingly, peptide-based neoantigen vaccine platforms have to date failed to consistently induce robust neoantigen-specific CD8 T cell responses in the majority of patients [13][14][15] . Although more immunogenic, a homologous prime boost messenger RNA (mRNA)-based vaccination approach elicited predominantly CD4 T cell responses [16][17][18] . Cumulatively, previous findings suggest that a successful cancer vaccine should (1) target tumor-specific neoantigens, (2) use highly immunogenic vaccine platform(s), (3) expand and prime T cells, (4) be combined with CPI therapy 19 and (5) generate long-term memory responses to ensure continuous tumor control for durable clinical benefit.Viral vector-based vaccine platforms, such as recombinant adenovirus, are able to prime robust T cell responses [20][21][22][23][24] . Although high seroprevalence of anti-adenoviral antibodies in human populations

No abstract

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell's microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2's cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell's microenvironment and may have implications in physical aspects of tumor biology.

Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.

The development of antimicrobial-resistant (AMR) bacteria poses a serious worldwide health concern. CRISPR-based antibacterials are a novel and adaptable method for building an arsenal of antibacterials potentially capable of targeting any pathogenic bacteria.

Miniaturization of lab-on-a-chip devices to nanoscale dimensions necessitates a level of systems integration currently found primarily in biological systems. Such devices will require new modes of transporting macromolecular materials at nanometer length scales. In cells, efficient cytoplasmic transport is achieved by energy-consuming, active transport systems in which motor proteins transport cargo along cytoskeletal filaments. For example, the motor protein kinesin-1 carries cell organelles and macromolecules over considerable distances along microtubule filaments. [1,2] Microtubules are hollow protein polymeric filaments with a diameter of %25 nm and tens of micrometers in length that form a 3D transportation network within the cell. Small groups of kinesin transport cargo at rates up to %12 mm s À1 , with a catalytic efficiency (i.e., conversion of chemical energy into work) of %50%. [3][4][5] Together, this transport system provides a highefficiency means of transporting macromolecular cargo through the highly viscous medium of cytoplasm.

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.