LIVE-PAINT: Super-Resolution Microscopy Inside Live Cells Using Reversible Peptide-Protein Interactions

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.

Intertwined gababutin-based supramolecular double helix

A dimer-assembly driven supramolecular double helix is observed for the gababutin-based short peptide sequence and this architecture exhibits electrochemical features.

Growth of Self-Assembled Bio-Organic Nanomatrices for Skin Tissue Engineering — An in vitro Study

In this paper, we have developed self-assembled nanoscale assemblies that were prepared by conjugating furan-2-carboxylic acid-3-aminopropyl amide with the short peptide sequence Gly-His (abbreviated Gly-His-FCAP). To mimic the extracellular matrix of mammalian fibroblasts and keratinocytes, the assemblies were then conjugated with Type I collagen. We then integrated the collagen bound Gly-His-FCAP assemblies with a short peptide sequence derived from salamander skin into the nanoscale assemblies for the first time to impart regenerative and wound healing properties to the composites. The antioxidant, antimicrobial and biodegradable properties were examined and results indicate that the nanocomposites displayed antioxidant properties as displayed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The biodegradability was found to be gradual. The nanocomposites were also found to inhibit the growth of the fungus Rhizopus sporangia over an 18[Formula: see text]h growth period. As proof of concept, to demonstrate the development of three-dimensional (3D) engineered skin in vitro, 3D printed PLA scaffolds of 2.5[Formula: see text]mm thickness were submerged in media containing nanocomposites and co-cultures of dermal fibroblasts with epidermal keratinocytes mimicking three dimensional skin substitute was examined. Our results indicated that the nanocomposites adhered to and supported cell proliferation and mimicked the components of skin and may have potential applications in skin tissue regeneration.

Integration of Biomolecules with Inorganic Ferroelectrics: A Novel Approach to Nanoscale Devices

ABSTRACTIn this work, we investigate the feasibility of using a surface-tethered heptapeptide sequence as the basis for a ferroelectric-actuated component in a nanofluidic device. The fluorescently-labeled peptide sequence, (CISLLHSTC) is shown by fluorescence microscopy to selectively coat the PZT patterned channel floors. The peptide binding strength to PZT is determined over a range of flow rates in the patterned channel by imaging the fluorescence intensity of the coated channel and monitoring the output spectroscopically. The peptide is found to be stripped from the PZT at flow rates exceeding 5mL/h. Initial results demonstrating the possibility of covalently integrating the short peptide sequence to larger biological components such as antibodies are also presented.

Selective Peptide Recognition With Molecularly Imprinted Polymers in Designing New Biomedical Devices

Molecular imprinting is a technique for the synthesis of polymers capable to bind selectively specific molecules. The imprinting of large proteins, like cell adhesion proteins or cell receptors, can lead to important and innovative biomedical applications. However such molecules show such important conformational changes in the polymerisation environment that the recognition sites are poorly specific. The “epitope approach” can overcome this limit by adopting, as template, a stable short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (macroporous monolith and nanospheres) were obtained with the protected peptides Z-Thr-Ala-Ala-OMe, as template, and Z-Thr-Ile-Leu-OMe, as analogue for the selectivity evaluation, the methacrylic acid, as functional monomer, the trimethylolpropane trimethacrylate and pentaerythritol triacrylate, as cross-linkers. Polymers were synthesized by precipitation polymerisation in acetonitrile at 60 °C, thermally initiated with azobisisobutyronitrile. All polymers were characterized by the standard techniques SEM, FT-IR, and TGA. The supernatants from the polymerisation and the rebinding solutions were analysed by HPLC. The higher cross-linked polymers retained about the 70% of the template, against about the 20% for the lower ones. The extracted template amount and the rebinding capacity decreased with the cross-linking degree, while the selectivity showed the opposite behaviour. The pentaerythritol triacrylate cross-linked polymers showed the best recognition (MIP 2−, α = 1.71) and selectivity (MIP 2+, α′ = 5.58) capabilities.

The Golgi sorting domain of coronavirus E1 protein

The coronavirus E1 membrane protein is confined to the Golgi after it is expressed in cells either by viral infection or via injection of synthetic RNA. We have investigated the features of the protein responsible for intracellular sorting and found that a C-terminal deletion of only 18 amino acids results in its transport to the plasma membrane. However, we have previously shown that this C-terminal region alone is not sufficient for Golgi retention. When E1 was fused to a cell-surface protein, Thy-1, the resulting molecule was retained in the Golgi. Various mutated forms of E1 whose destinations were the ER, cell surface or lysosomes were also fused to Thy-1, and in each case the fusion was sorted according to its E1 component alone. We argue that, in contrast to sorting signals for other membrane compartments, Golgi retention of E1 is not due to a single short peptide sequence. Instead, the Golgi ‘signal’ of E1 appears to require for its expression a domain comprising most of the sequence of the protein.