Disulfide-Bond Scrambling Promotes Amorphous Aggregates in Lysozyme and Bovine Serum Albumin

Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris­(2-carboxyethyl)­phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins

Self-repairing design process applied to a 4-bar linkage mechanism

Despite significant advances in modelling and design, mechanical systems almost inevitably fail at some point during their operative life. This can be due to a pre-existing design flaw, which is usually overcome in a revision, or more commonly due to some unexpected damage during operation. To overcome a failure during operation, a new method of designing machines or systems is proposed that creates a result that is resilient to both expected and unexpected failure. By shifting the focus from a detailed assessment of the underlying cause of failure to how that failure will manifest, a system becomes inherently resilient against a wide range of failure modes. The proposed process involves five steps: Cause, Detection, Diagnosis, Confirmation, and Correction. This is demonstrated with an application to a generic four-bar linkage mechanism. Through this process the system is able to return to a near perfect state even after a permanent deformation occurs in the mechanism. These results show the potential that this self-repairing design process has for applications including robotics, manufacturing and other systems

Preformed Seeds Modulate Native Insulin Aggregation Kinetics

Insulin aggregates under storage conditions via disulfide interchange reaction. It is also known to form aggregates at the site of repeated injections in diabetes patients, leading to injection amyloidosis. This has fueled research in pharmaceutical and biotechnology industry as well as in academia to understand factors that modulate insulin stability and aggregation. The main aim of this study is to understand the factors that modulate aggregation propensity of insulin under conditions close to physiological and measure effect of “<i>seeds</i>” on aggregation kinetics. We explored the aggregation kinetics of insulin at pH 7.2 and 37 °C in the presence of disulfide-reducing agent dithiothreitol (DTT), using spectroscopy (UV–visible, fluorescence, and Fourier transform infrared spectroscopy) and microscopy (scanning electron microscopy, atomic force microscopy) techniques. We prepared insulin “<i>seeds</i>” by incubating disulfide-reduced insulin at pH 7.2 and 37 °C for varying lengths of time (10 min to 12 h). These seeds were added to the native protein and nucleation-dependent aggregation kinetics was measured. Aggregation kinetics was fastest in the presence of 10 min seeds suggesting they were <i>nascent.</i> Interestingly, <i>intermediate</i> seeds (30 min to 4 h incubation) resulted in formation of transient fibrils in 4 h that converted to amorphous aggregates upon longer incubation of 24 h. Overall, the results show that insulin under disulfide reducing conditions at pH and temperature close to physiological favors amorphous aggregate formation and seed “maturity” plays an important role in nucleation dependent aggregation kinetics

Studies on Bacterial Proteins Corona Interaction with Saponin Imprinted ZnO Nanohoneycombs and Their Toxic Responses

Molecular imprinting generates robust, efficient, and highly mesoporous surfaces for biointeractions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understand the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesized saponin imprinted zinc oxide nanohoneycombs (SIZnO NHs), average size 80–125 nm, surface area 20.27 m²/g, average pore density 0.23 pore/nm and number-average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential antifungal and antibacterial agents have been studied on <i>Sclerotium rolfsii (S. rolfsii)</i>, <i>Pythium debarynum (P. debarynum) and Escherichia coli (E. coli)</i>, <i>Staphylococcus aureus (S. aureus)</i>, respectively. SIZnO NHs exhibited the highest antibacterial (∼50%) and antifungal (∼40%) activity against Gram-negative bacteria (<i>E. coli</i>) and fungus (<i>P. debarynum)</i>, respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalized into the cell. The molecular docking studies were carried out using binding proteins present in the gram negative bacteria (<i>lipopolysaccharide</i> and <i>lipocalin Blc</i>) and gram positive bacteria (Staphylococcal Protein A, SpA). It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies were higher in the case of binding proteins present in gram negative bacteria as compared to that of gram positive bacteria. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalization in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good agreement with the experimental data. Exploration of mechanistic interactions, as well as the formation of bioconjugate of proteins and ZnO NHs would play a key role to interpret more complex biological systems in nature

Switchable Bioelectrocatalysis Controlled by Dual Stimuli-Responsive Polymeric Interface

The engineering of bionanointerfaces using stimuli-responsive polymers offers a new dimension in the design of novel bioelectronic interfaces. The integration of electrode surfaces with stimuli-responsive molecular cues provides a direct control and ability to switch and tune physical and chemical properties of bioelectronic interfaces in various biodevices. Here, we report a dual-responsive biointerface employing a positively responding dual-switchable polymer, poly­(NIPAAm-<i>co</i>-DEAEMA)-<i>b-</i>HEAAm, to control and regulate enzyme-based bioelectrocatalysis. The design interface exhibits reversible activation–deactivation of bioelectrocatalytic reactions in response to change in temperature and in pH, which allows manipulation of biomolecular interactions to produce on/off switchable conditions. Using electrochemical measurements, we demonstrate that interfacial bioelectrochemical properties can be tuned over a modest range of temperature (i.e., 20–60 °C) and pH (i.e., pH 4–8) of the medium. The resulting dual-switchable interface may have important implications not only for the design of responsive biocatalysis and on-demand operation of biosensors, but also as an aid to elucidating electron-transport pathways and mechanisms in living organisms by mimicking the dynamic properties of complex biological environments and processes

Ultrasensitive Detection of Human Liver Hepatocellular Carcinoma Cells Using a Label-Free Aptasensor

Liver cancer is one of the most common cancers in the world and has no effective cure, especially in later stages. The development of a tangible protocol for early diagnosis of this disease remains a major challenge. In the present manuscript, an aptamer-based, label-free electrochemical biosensor for the sensitive detection of HepG2, a hepatocellular carcinoma cell line, is described. The target cells are captured in a sandwich architecture using TLS11a aptamer covalently attached to a gold surface and a secondary TLS11a aptamer. The application of TLS11a aptamer as a recognition layer resulted in a sensor with high affinity for HepG2 cancer cells in comparison with control cancer cells of human prostate, breast, and colon tumors. The aptasensor delivered a wide linear dynamic range over 1 × 10² to 1 × 10⁶ cells/mL, with a detection limit of 2 cells/mL. This protocol provides a precise method for sensitive detection of liver cancer with significant advantages in terms of simplicity, low cost, and stability

Self-Reporting Micellar Polymer Nanostructures for Optical Urea Biosensing

We report the facile fabrication of a self-reporting, highly sensitive and selective optical urea nanobiosensor using chitosan-<i>g</i>-polypyrrole (CHIT-<i>g</i>-PPy) nanomicelles as a sensing platform. Urease was immobilized on the spherical micellar surface to create an ultrasensitive self-reporting nanobiosystem for urea. The resulting nanostructures show monodisperse size distributions before and after enzyme loading. The critical micelle concentration of the enzyme-immobilized polymer nanostructure was measured to be 0.49 mg/L in phosphate buffer at pH 7.4. The nanobiosensor had a linear optical response to urea concentrations ranging from 0.01 to 30 mM with a response time of a few seconds. This promising approach provides a novel methodology for self-reporting bioassembly over nanostructure polymer micelles and furnishes the basis for fabrication of sensitive and efficient optical nanobiosensors

Unusual Fluorescent Responses of Morpholine-Functionalized Fluorescent Probes to pH via Manipulation of BODIPY’s HOMO and LUMO Energy Orbitals for Intracellular pH Detection

Three uncommon morpholine-based fluorescent probes (<b>A</b>, <b>B</b>, and <b>C</b>) for pH were prepared by introducing morpholine residues to BODIPY dyes at 4,4′- and 2,6-positions, respectively. In contrast to morpholine-based fluorescent probes for pH reported in literature, these fluorescent probes display high fluorescence in a basic condition while they exhibit very weak fluorescence in an acidic condition. The theoretical calculation confirmed that morpholine is unable to function as either an electron donor or an electron acceptor to quench the BODIPY fluorescence in the neutral and basic condition via photoinduced electron transfer (PET) mechanism because the LUMO energy of morpholine is higher than those of the BODIPY dyes while its HOMO energy is lower than those of the BODIPY dyes. However, the protonation of tertiary amines of the morpholine residues in an acidic environment leads to fluorescence quenching of the BODIPY dyes via d-PET mechanism. The fluorescence quenching is because the protonation effectively decreases the LUMO energy which locates between the HOMO and LUMO energies of the BODIPY dyes. Fluorescent probe <b>C</b> with deep-red emission has been successfully used to detect pH changes in mammalian cells

Near-Infrared Fluorescent Probes with Large Stokes Shifts for Sensing Zn(II) Ions in Living Cells

We report two new near-infrared fluorescent probes based on Rhodol counterpart fluorophore platforms functionalized with dipicolylamine Zn­(II)-binding groups. The combinations of the pendant amines and fluorophores provide the probes with an effective three-nitrogen-atom and one-oxygen-atom binding motif. The fluorescent probes with large Stokes shifts offer sensitive and selective florescent responses to Zn­(II) ions over other metal ions, allowing a reversible monitoring of Zn­(II) concentration changes in living cells, and detecting intracellular Zn­(II) ions released from intracellular metalloproteins

Pseudophosphorylation of kinesin-1 at S175/S176 inhibits movement of kinesin-1.

<p>To determine the effects of modifying S175 and S176 on kinesin-1function, recombinant GFP-tagged kinesin (KHC⁵⁵⁹) was modified to preclude phosphorylation at these sites (S175AS176A) or to mimic phosphorylation (S175ES176E). <b>(a–f)</b> Stage 3 hippocampal neurons were examined 5 h after co-transfection with GFP-tagged KHC⁵⁵⁹ constructs and a tdTomato construct, which diffuses throughout the cell and allows visualization of neurites (<b>b, d, f</b>). Both wild-type kinesin-1 (KHC⁵⁵⁹ WT, <b>a</b>) and a non-phosphorylatable mutant (KHC⁵⁵⁹ S175A/S176A, <b>c</b>) accumulated efficiently at axonal tips (labeled by arrows) with minimal steady-state labeling of cell bodies (arrowheads). In contrast, pseudophosphorylated mutant KHC⁵⁵⁹ S175E/S176E, <b>e</b>) was mainly present in neuronal cell bodies. Quantitative immunofluorescence analysis shows fraction of total KHC⁵⁵⁹ fluorescence at axon tips for all constructs <b>(g)</b>. Far less phosphomimicking KHC⁵⁵⁹ S175E/S176E constructs accumulated at axon tips than KHC⁵⁵⁹ WT or KHC⁵⁵⁹ S175A/S176A (#: p<0.001; <i>n</i>: 27–43 cells per condition). Bars show mean and standard deviation. Scale bar  = 20 µm.</p