Interrogating Matrix Stiffness and Metabolomics in Pancreatic Ductal Carcinoma Using an Openable Microfluidic Tumor-on-a-Chip.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma that contributes to aggressive tumor biology and therapeutic resistance. Current in vitro PDAC models lack sufficient optical and physical access for fibrous network visualization, in situ mechanical stiffness measurement, and metabolomic profiling. Here, we describe an openable multilayer microfluidic PDAC-on-a-chip platform that consists of pancreatic tumor cells (PTCs) and pancreatic stellate cells (PSCs) embedded in a 3D collagen matrix that mimics the stroma. Our system allows fibrous network visualization via reflected light confocal (RLC) microscopy, in situ mechanical stiffness testing using atomic force microscopy (AFM), and compartmentalized hydrogel extraction for PSC metabolomic profiling via mass spectrometry (MS) analysis. In comparing cocultures of gel-embedded PSCs and PTCs with PSC-only monocultures, RLC microscopy identified a significant decrease in pore size and corresponding increase in fiber density. In situ AFM indicated significant increases in stiffness, and hallmark characteristics of PSC activation were observed using fluorescence microscopy. PSCs in coculture also demonstrated localized fiber alignment and densification as well as increased collagen production. Finally, an untargeted MS study putatively identified metabolic contributions consistent with in vivo PDAC studies. Taken together, this platform can potentially advance our understanding of tumor-stromal interactions toward the discovery of novel therapies.

Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

TANDEM: biomicrofluidic systems with transverse and normal diffusional environments for multidirectional signaling.

Biomicrofluidic systems that can recapitulate complex biological processes with precisely controlled 3D geometries are a significant advancement from traditional 2D cultures. To this point, these systems have largely been limited to either laterally adjacent channels in a single plane or vertically stacked single-channel arrangements. As a result, lateral (or transverse) and vertical (or normal) diffusion have been isolated to their respective designs only, thus limiting potential access to nutrients and 3D communication that typifies in vivo microenvironments. Here we report a novel device architecture called "TANDEM", an acronym for "T̲ransverse A̲nd N̲ormal D̲iffusional E̲nvironments for M̲ultidirectional Signaling", which enables multiplanar arrangements of aligned channels where normal and transverse diffusion occur in tandem to facilitate multidirectional communication. We developed a computational transport model in COMSOL and tested diffusion and culture viability in one specific TANDEM configuration, and found that TANDEM systems demonstrated enhanced diffusion in comparison to single-plane counterparts. This resulted in improved viability of hydrogel-embedded cells, which typically suffer from a lack of sufficient nutrient access during long-term culture. Finally, we showed that TANDEM designs can be expanded to more complex alternative configurations depending on the needs of the end-user. Based on these findings, TANDEM designs can utilize multidirectional enhanced diffusion to improve long-term viability and ultimately facilitate more robust and more biomimetic microfluidic systems with increasingly more complex geometric layouts.

Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of de novo telomere addition.

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.

Integrated electrochemical measurement of endothelial permeability in a 3D hydrogel-based microfluidic vascular model.

Mimicking the physiological or pathophysiological barrier function of endothelial and epithelial cells is an essential consideration in organ-on-a-chip models of numerous tissues including the vascular system, lungs, gut and blood-brain barrier. Recent models have furthermore incorporated 3D extracellular matrix hydrogels to recapitulate the composition and cell-matrix interactions found in the native microenvironment. Assessment of barrier function in these 3D organ-on-a-chip models, however, is typically limited to diffusive permeability measurements that are exclusively fluorescence-based. In this work, an on-chip electrochemical method to measure endothelial permeability in a 3D hydrogel-based vascular model was developed that replaces the ubiquitous fluorescent tracer with an electroactive one. Unlike the traditional fluorescent-based method, this electrochemical method eliminates the need for bulky, costly and complex optical instrumentation that require measurements to be performed outside of the incubator. A 3D extracellular matrix gel-based microfluidic model was first developed that incorporates capillary pressure barrier microstructures. Micromilling of thermoplastics was used to fabricate these microstructures in a rapid, moldless fashion. As a proof-of-concept demonstration, the permeability of endothelial cells cultured on hydrogels was electrochemically measured after being subject to perfusion conditions, and following exposure to known permeability mediators. In summary, the electrochemical permeability assay possesses both the benefits of on-chip integration and robustness of the traditional fluorescence-based assay while also enabling the measurement of barrier function in an organ-on-a-chip incorporating 3D culture conditions.

Amyloid-ß(1-42) protofibrils stimulate a quantum of secreted IL-1ß despite significant intracellular IL-1ß accumulation in microglia.

Neuroinflammation is a characteristic feature of the Alzheimer's disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-ß protein (Aß). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aß plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aß(1-42) protofibrils. Aß(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1ß (IL-1ß) mRNA, and intracellular pro and mature forms of IL-1ß protein. The accumulation of both IL-1ß forms indicated that Aß(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aß-induced accumulation of intracellular mature IL-1ß did not translate into greater IL-1ß secretion. Instead, we found that Aß elicited a quantized burst of secreted IL-1ß and this process occurred even prior to Aß priming of the microglia suggesting a basal level of either pro or mature IL-1ß in the cultured primary microglia. The IL-1ß secretion burst was rapid but not sustained, yet could be re-evoked with additional Aß stimulation. The findings from this study demonstrated multiple sites of IL-1ß regulation by Aß(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1ß secretory process. These results underscore the wide-ranging effects of Aß on the innate immune response.

The crystal structure of Bacillus subtilis YycI reveals a common fold for two members of an unusual class of sensor histidine kinase regulatory proteins.

YycI and YycH are two membrane-anchored periplasmic proteins that regulate the essential Bacillus subtilis YycG histidine kinase through direct interaction. Here we present the crystal structure of YycI at a 2.9-A resolution. YycI forms a dimer, and remarkably the structure resembles that of the two C-terminal domains of YycH despite nearly undetectable sequence homology (10%) between the two proteins.

YycH and YycI interact to regulate the essential YycFG two-component system in Bacillus subtilis.

The YycFG two-component system is the only signal transduction system in Bacillus subtilis known to be essential for cell viability. This system is highly conserved in low-G+C gram-positive bacteria, regulating important processes such as cell wall homeostasis, cell membrane integrity, and cell division. Four other genes, yycHIJK, are organized within the same operon with yycF and yycG in B. subtilis. Recently, it was shown that the product of one of these genes, the YycH protein, regulated the activity of this signal transduction system, whereas no function could be assigned to the other genes. Results presented here show that YycI and YycH proteins interact to control the activity of the YycG kinase. Strains carrying individual in-frame deletion of the yycI and yycH coding sequences were constructed and showed identical phenotypes, namely a 10-fold-elevated expression of the YycF-dependent gene yocH, growth defects, as well as a cell wall defect. Cell wall and growth defects were a direct result of overregulation of the YycF regulon, since a strain overexpressing YycF showed phenotypes similar to those of yycH and yycI deletion strains. Both YycI and YycH proteins are localized outside the cytoplasm and attached to the membrane by an N-terminal transmembrane sequence. Bacterial two-hybrid data showed that the YycH, YycI, and the kinase YycG form a ternary complex. The data suggest that YycH and YycI control the activity of YycG in the periplasm and that this control is crucial in regulating important cellular processes.

The crystal structure of YycH involved in the regulation of the essential YycFG two-component system in Bacillus subtilis reveals a novel tertiary structure.

The Bacillus subtilis YycFG two-component signal transduction system is essential for cell viability, and the YycH protein is part of the regulatory circuit that controls its activity. The crystal structure of YycH was solved by two-wavelength selenium anomalous dispersion data, and was refined using 2.3 A data to an R-factor of 25.2%. The molecule is made up of three domains, and has a novel three-dimensional structure. The N-terminal domain features a calcium binding site and the central domain contains two conserved loop regions.

Two-dimensional diversity: screening human cDNA phage display libraries with a random diversity probe for the display cloning of phosphotyrosine binding domains.

A random phosphopeptide probe (bio-pYZZZ) has been used for the isolation and identification of multiple SH2 domains from human cDNA-displaying phage libraries. In addition, on-phage analysis and quantification of binding affinities for these phage-displayed proteins has shown them to be functional domains, retaining the same characteristics as in their native state.