Integrative Click Chemistry for Tuning Physicochemical Properties of Cancer Cell-Laden Hydrogels

Indiana University-Purdue University Indianapolis (IUPUI)The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix sti ness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modi ed and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix sti ness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix sti ening and increased HA presentation might a ect survival of PDAC cells and their response to chemotherapeutics

Quantifying physiological influences on otolith microchemistry

Trace element concentrations in fish earstones (‘otoliths’) are widely used to discriminate spatially discrete populations or individuals of marine fish, based on a commonly held assumption that physiological influences on otolith composition are minor, and thus variations in otolith elemental chemistry primarily reflect changes in ambient water chemistry. We carried out a long-term (1-year) experiment, serially sampling seawater, blood plasma and otoliths of mature and immature European plaice (Pleuronectes platessa L.) to test relationships between otolith chemistry and environmental and physiological variables. Seasonal variations in otolith elemental composition did not track seawater concentrations, but instead reflected physiological controls on metal transport and biokinetics, which are likely moderated by ambient temperature. The influence of physiological factors on otolith composition was particularly evident in Sr/Ca ratios, the most widely used elemental marker in applied otolith microchemistry studies. Reproduction also triggered specific variations in otolith and blood plasma metal chemistry, especially Zn/Ca ratios in female fish, which could potentially serve as retrospective spawning indicators. The influence of physiology on the trace metal composition of otoliths may explain the success of microchemical stock discrimination in relatively homogenous marine environments, but could complicate alternative uses for trace element compositions in biominerals of higher organism

The Shape of LITTLE THINGS Dwarf Galaxies DDO 46 and DDO 168: Understanding the stellar and gas kinematics

We present the stellar and gas kinematics of DDO 46 and DDO 168 from the LITTLE THINGS survey and determine their respective Vmax/sigma_z,0 values. We used the KPNO's 4-meter telescope with the Echelle spectrograph as a long-slit spectrograph. We acquired spectra of DDO 168 along four position angles by placing the slit over the morphological major and minor axes and two intermediate position angles. However, due to poor weather conditions during our observing run for DDO 46, we were able to extract only one useful data point from the morphological major axis. We determined a central stellar velocity dispersion perpendicular to the disk, sigma_z,0, of 13.5+/-8 km/s for DDO 46 and of 10.7+/-2.9 km/s for DDO 168. We then derived the maximum rotation speed in both galaxies using the LITTLE THINGS HI data. We separated bulk motions from non-circular motions using a double Gaussian decomposition technique and applied a tilted-ring model to the bulk velocity field. We corrected the observed HI rotation speeds for asymmetric drift and found a maximum velocity, Vmax, of 77.4 +/- 3.7 and 67.4 +/- 4.0 km/s for DDO 46 and DDO 168, respectively. Thus, we derived a kinematic measure, Vmax/sigma_z,0, of 5.7 +/- 0.6 for DDO 46 and 6.3 +/- 0.3 for DDO 168. Comparing these values to ones determined for spiral galaxies, we find that DDO 46 and DDO 168 have Vmax/sigma_z,0 values indicative of thin disks, which is in contrast to minor-to-major axis ratio studies

Single-molecule study of redox control involved in establishing the spinach plastocyanin-cytochrome b6f electron transfer complex

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET

Correlated fluorescence quenching and topographic mapping of Light-Harvesting Complex II within surface-assembled aggregates and lipid bilayers

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation

Correlated fluorescence quenching and topographic mapping of Light-Harvesting Complex II within surface-assembled aggregates and lipid bilayers

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation

Nanodomains of Cytochrome b(6)f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b6f (cytb6f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb6f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb6f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb6f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb6f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb6f complexes. We suggest that the close proximity between PSII and cytb6f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency

Interference lithographic nanopatterning of plant and bacterial light-harvesting complexes on gold substrates

We describe a facile approach for nanopatterning of photosynthetic light-harvesting complexes over macroscopic areas, and use optical spectroscopy to demonstrate retention of native properties by both site-specifically and non-specifically attached photosynthetic membrane proteins. A Lloyd's mirror dual-beam interferometer was used to expose self-assembled monolayers of amine-terminated alkylthiolates on gold to laser irradiation. Following exposure, photo-oxidized adsorbates were replaced by oligo(ethylene glycol) terminated thiols, and the remaining intact amine-functionalized regions were used for attachment of the major light-harvesting chlorophyll–protein complex from plants, LHCII. These amine patterns could be derivatized with nitrilotriacetic acid (NTA), so that polyhistidine-tagged bacteriochlorophyll–protein complexes from phototrophic bacteria could be attached with a defined surface orientation. By varying parameters such as the angle between the interfering beams and the laser irradiation dose, it was possible to vary the period and widths of NTA and amine-functionalized lines on the surfaces; periods varied from 1200 to 240 nm and linewidths as small as 60 nm (λ/4) were achieved. This level of control over the surface chemistry was reflected in the surface topology of the protein nanostructures imaged by atomic force microscopy; fluorescence imaging and spectral measurements demonstrated that the surface-attached proteins had retained their native functionality