Non-Covalently Associated Streptavidin Multi-Arm Nanohubs Exhibit Mechanical and Thermal Stability in Cross-Linked Protein-Network Materials.

Constructing protein-network materials that exhibit physicochemical and mechanical properties of individual protein constituents requires molecular cross-linkers with specificity and stability. A well-known example involves specific chemical fusion of a four-arm polyethylene glycol (tetra-PEG) to desired proteins with secondary cross-linkers. However, it is necessary to investigate tetra-PEG-like biomolecular cross-linkers that are genetically fused to the proteins, simplifying synthesis by removing additional conjugation and purification steps. Non-covalently, self-associating, streptavidin homotetramer is a viable, biomolecular alternative to tetra-PEG. Here, a multi-arm streptavidin design is characterized as a protein-network material platform using various secondary, biomolecular cross-linkers, such as high-affinity physical (i.e., non-covalent), transient physical, spontaneous chemical (i.e., covalent), or stimuli-induced chemical cross-linkers. Stimuli-induced, chemical cross-linkers fused to multi-arm streptavidin nanohubs provide sufficient diffusion prior to initiating permanent covalent bonds, allowing proper characterization of streptavidin nanohubs. Surprisingly, non-covalently associated streptavidin nanohubs exhibit extreme stability, which translates into material properties that resemble hydrogels formed by chemical bonds even at high temperatures. Therefore, this study not only establishes that the streptavidin nanohub is an ideal multi-arm biopolymer precursor but also provides valuable guidance for designing self-assembling nanostructured molecular networks that can properly harness the extraordinary properties of protein-based building blocks.

Layered supramolecular hydrogels from thioglycosides.

Low molecular weight hydrogels are made of small molecules that aggregate via noncovalent interactions. Here, comprehensive characterization of the physical and chemical properties of hydrogels made from thioglycolipids of the disaccharides lactose and cellobiose with simple alkyl chains is reported. While thiolactoside hydrogels are robust, thiocellobioside gels are metastable, precipitating over time into fibrous crystals that can be entangled to create pseudo-hydrogels. Rheology confirms the viscoelastic solid nature of these hydrogels with storage moduli ranging from 10-600 kPa. Additionally, thiolactoside hydrogels are thixotropic which is a desirable property for many potential applications. Freeze-fracture electron microscopy of xerogels shows layers of stacked sheets that are entangled into networks. These structures are unique compared to the fibers or ribbons typically reported for hydrogels. Differential scanning calorimetry provides gel-to-liquid phase transition temperatures ranging from 30 to 80 °C. Prodan fluorescence spectroscopy allows assignment of phase transitions in the gels and other lyotropic phases of high concentration samples. Phase diagrams are estimated for all hydrogels at 1-10 wt% from 5 to ≥ 80 °C. These hydrogels represent a series of interesting materials with unique properties that make them attractive for numerous potential applications.

Alteration of fibrin hydrogel gelation and degradation kinetics through addition of azo dyes.

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength.

Cytoskeleton-inspired artificial protein design to enhance polymer network elasticity.

Reducing topological network defects to enhance elasticity in polymeric materials remains a grand challenge. Efforts to control network topology, primarily focused on crosslinking junctions, continue to underperform compared to theoretical estimations from idealized networks using affine and phantom network theories. Here, artificial protein technology was adapted for the design of polymer-network hydrogels with precisely defined coil-like and rod-like strands to observe the impact of strand rigidity on the mechanical properties of polymeric materials. Cytoskeleton-inspired polymer-network hydrogels incorporated with rod-like protein strands nearly tripled the gel shear elastic modulus and relaxation time compared to coil-like protein strands, indicating an enhanced effective crosslinking density. Furthermore, asymmetric rod-coil protein designs in network strands with an optimal rod:coil ratio improved the hydrogel relaxation time, enhancing the stability of physical macromolecular associations by modulating crosslinker mobility. The careful design of strand rigidity presents a new direction to reduce topological defects for optimizing polymeric materials.

Non-cytotoxic Dityrosine Photocrosslinked Polymeric Materials With Targeted Elastic Moduli.

Controlling mechanical properties of polymeric biomaterials, including the elastic modulus, is critical to direct cell behavior, such as proliferation and differentiation. Dityrosine photocrosslinking is an attractive and simple method to prepare materials that exhibit a wide range of elastic moduli by rapidly crosslinking tyrosyl-containing polymers. However, high concentrations of commonly used oxidative crosslinking reagents, such as ruthenium-based photoinitiators and persulfates, present cytotoxicity concerns. We found the elastic moduli of materials prepared by crosslinking an artificial protein with tightly controlled tyrosine molarity can be modulated up to 40 kPa by adjusting photoinitiator and persulfate concentrations. Formulations with various concentrations of the crosslinking reagents were able to target a similar material elastic modulus, but excess unreacted persulfate resulted in cytotoxic materials. Therefore, we identified a systematic method to prepare non-cytotoxic photocrosslinked polymeric materials with targeted elastic moduli for potential biomaterials applications in diverse fields, including tissue engineering and 3D bioprinting.

Smartphone based on-chip fluorescence imaging and capillary flow velocity measurement for detecting ROR1+ cancer cells from buffy coat blood samples on dual-layer paper microfluidic chip.

Diagnosis of hematological cancer requires complete white blood cell count, followed by flow cytometry with multiple markers, and cytology. It requires substantial time and specialized training. A dual-layer paper microfluidic chip was developed as a quicker, low-cost, and field-deployable alternative to detect ROR1+ (receptor tyrosine-like orphan receptor one) cancer cells from the undiluted and untreated buffy coat blood samples. The first capture layer consisted of a GF/D glass fiber substrate, preloaded with cancer specific anti-ROR1 conjugated fluorescent particles to its center for cancer cell capture and direct smartphone fluorescence imaging. The second flow layer was comprised of a grade 1 cellulose chromatography paper with wax-printed four channels for wicking and capillary flow-based detection. The flow velocity was used as measure of antigen concentration in the buffy coat sample. In this manner, intact cells and their antigens were separated and independently analyzed by both imaging and flow velocity analyses. A custom-made smartphone-based fluorescence microscope and automated image processing and particle counter software were developed to enumerate particles on paper, with the limit of detection of 1 cell/µL. Flow velocity analysis showed even greater sensitivity, with the limit of detection of 0.1 cells/µL in the first 6 s of assay. Comparison with capillary flow model revealed great alignment with experimental data and greater correlation to viscosity than interfacial tension. Our proposed device is able to capture and on-chip image ROR1+ cancer cells within a complex sample matrix (buffy coat) while simultaneously quantifying cell concentration in a point-of-care manner.

Liquid Amphiphilic Polymer for Effective Airborne Dust Suppression.

Airborne dust is a byproduct of natural and artificial occurrences, including high winds in arid regions and human activities that affect most of the world's population. Watering is the most general practice for reducing airborne dust by wetting the surface of the dust source to agglomerate dust particles via capillary effect, increasing the aerodynamic diameter of (ultra)fine particles and reducing dust emission. However, the short-term effectiveness due to fast water evaporation, requiring frequent watering, is a major disadvantage. Herein, we utilized biocompatible liquid polymers as additives in water to prolong moist conditions of dust sources due to their liquid state. After the water evaporated, liquid polymers maintained moisture on dust sources, resulting in significantly reduced (ultra)fine particle emissions and extended effectiveness compared to conventional water treatment. Interestingly, we observed greater dust suppressive effectiveness with liquid amphiphilic polymer than liquid hydrophilic polymer because of the synergistic effect of the liquid state and amphiphilic property of the polymer. Translating lab-scale experiments to pilot-scale field-testing confirmed the potential for utilizing biocompatible liquid amphiphilic polymers to advance airborne dust suppression technology.

Drug sensitivity of single cancer cells is predicted by changes in mass accumulation rate.

Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, the utility of current functional assays is limited, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. We found that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defined the drug sensitivity or resistance of glioblastoma and B-cell acute lymphocytic leukemia cells. MAR revealed heterogeneity in drug sensitivity not only between different tumors, but also within individual tumors and tumor-derived cell lines. MAR measurement predicted drug response using samples as small as 25 µl of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance in heterogeneous tumors.

MAPK activation and HRAS mutation identified in pituitary spindle cell oncocytoma.

Pituitary spindle cell oncocytoma (SCO) is an uncommon primary pituitary neoplasm that presents with mass effect on adjacent neurovascular structures, similar to non-hormone-producing pituitary adenomas. To determine the molecular etiology of SCO, we performed exome sequencing on four SCO cases, with matched normal controls, to assess somatic mutations and copy number alterations. Our analysis revealed a low mutation rate and a copy-neutral profile, consistent with the low-grade nature of this tumor. However, we identified a co-occurring somatic HRAS (p.Q61R) activating point mutation and MEN1 frameshift mutation (p.L117fs) present in a primary and recurrent tumor from one patient. Other SCOs demonstrated mutations in SND1 and FAT1, which are associated with MAPK pathway activation. Immunohistochemistry across the SCO cohort demonstrated robust MAPK activity in all cases (n=4), as evidenced by strong phospho-ERK staining, while phospho-AKT levels suggested only basal levels of PI3K pathway activation. Taken together, this identifies the MAPK signaling pathway as a novel therapeutic target for spindle cell oncocytoma, which may offer a powerful adjunct for aggressive tumors refractory to surgical resection.

Preclinical antitumor efficacy of selective exportin 1 inhibitors in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is poorly responsive to current chemotherapy. The nuclear transporter exportin 1 (XPO1, CRM1) is often highly expressed in GBM, which may portend a poor prognosis. Here, we determine the efficacy of novel selective inhibitors of nuclear export (SINE) specific to XPO1 in preclinical models of GBM. METHODS: Seven patient-derived GBM lines were treated with 3 SINE compounds (KPT-251, KPT-276, and Selinexor) in neurosphere culture conditions. KPT-276 and Selinexor were also evaluated in a murine orthotopic patient-derived xenograft (PDX) model of GBM. Cell cycle effects were assayed by flow cytometry in vitro and immunohistochemistry in vivo. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase 3/7 activity assays. RESULTS: Treatment of GBM neurosphere cultures with KPT-276, Selinexor, and KPT-251 revealed dose-responsive growth inhibition in all 7 GBM lines [range of half-maximal inhibitory concentration (IC50), 6-354 nM]. In an orthotopic PDX model, treatment with KPT-276 and Selinexor demonstrated pharmacodynamic efficacy, significantly suppressed tumor growth, and prolonged animal survival. Cellular proliferation was not altered with SINE treatment. Instead, induction of apoptosis was apparent both in vitro and in vivo with SINE treatment, without overt evidence of neurotoxicity. CONCLUSIONS: SINE compounds show preclinical efficacy utilizing in vitro and in vivo models of GBM, with induction of apoptosis as the mechanism of action. Selinexor is now in early clinical trials in solid and hematological malignancies. Based on these preclinical data and excellent brain penetration, we have initiated clinical trials of Selinexor in patients with relapsed GBM.

Clinical multiplexed exome sequencing distinguishes adult oligodendroglial neoplasms from astrocytic and mixed lineage gliomas.

Classifying adult gliomas remains largely a histologic diagnosis based on morphology; however astrocytic, oligodendroglial and mixed lineage tumors can display overlapping histologic features. We used multiplexed exome sequencing (OncoPanel) on 108 primary or recurrent adult gliomas, comprising 65 oligodendrogliomas, 28 astrocytomas and 15 mixed oligoastrocytomas to identify lesions that could enhance lineage classification. Mutations in TP53 (20/28, 71%) and ATRX (15/28, 54%) were enriched in astrocytic tumors compared to oligodendroglial tumors of which 4/65 (6%) had mutations in TP53 and 2/65 (3%) had ATRX mutations. We found that oligoastrocytomas harbored mutations in TP53 (80%, 12/15) and ATRX (60%, 9/15) at frequencies similar to pure astrocytic tumors, suggesting that oligoastrocytomas and astrocytomas may represent a single genetic or biological entity. p53 protein expression correlated with mutation status and showed significant increases in astrocytomas and oligoastrocytomas compared to oligodendrogliomas, a finding that also may facilitate accurate classification. Furthermore our OncoPanel analysis revealed that 15% of IDH1/2 mutant gliomas would not be detected by traditional IDH1 (p.R132H) antibody testing, supporting the use of genomic technologies in providing clinically relevant data. In all, our results demonstrate that multiplexed exome sequencing can support evaluation and classification of adult low-grade gliomas with a single clinical test.