Self-Assembling Coiled-Coil Peptide Nanotubes with Biomolecular Cargo Encapsulation.

We report a coiled-coil trimeric peptide based on the known GCN4 zipper motif that self-assembles into nanotubes with diameters on the order of a few hundred nanometers to a micron. The dimensional morphology of these tubular structures was observed to be tunable by altering the properties of the buffer during assembly. Structural evidence from X-ray scattering and electron microscopy suggest that tube assembly takes place in structural tiers leading to a hexagonal close-packed arrangement of the coiled-coils. The hollow tubes were observed to selectively encapsulate fluorescein-labeled anionic dextran, as a result of electrostatic interactions between the inner tube surface and negatively charged cargo. The ability of these nanotubes to house biomolecular cargo endows them with potential for molecular storage and drug delivery applications.

A Library Approach to Cationic Amphiphilic Polyproline Helices that Target Intracellular Pathogenic Bacteria.

A number of pathogenic bacteria reproduce inside mammalian cells and are thus inaccessible to many antimicrobial drugs. Herein, we present a facile method to a focused library of antibacterial agents known as cationic amphiphilic polyproline helices (CAPHs). We identified three CAPHs from the library with superior cell penetration within macrophages and excellent antibacterial action against both Gram-positive and Gram-negative bacteria. These cell-penetrating antibacterial CAPHs have specific subcellular localizations that allow for targeting of pathogenic bacteria at their intracellular niches, a unique feature that promotes the successful clearance of intracellular pathogens ( Salmonella, Shigella, and Listeria) residing within macrophages. Furthermore, the selected CAPHs also significantly reduced bacterial infections in an in vivo model of Caenorhabditis elegans, with minimal in vivo toxicity.

Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly.

Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design.

Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

Antibacterial activity and therapeutic efficacy of Fl-P(R)P(R)P(L)-5, a cationic amphiphilic polyproline helix, in a mouse model of staphylococcal skin infection.

The antibacterial activities and therapeutic efficacy of the cationic, unnatural proline-rich peptide Fl-P(R)P(R)P(L)-5 were evaluated against multidrug-resistant Staphylococcus aureus in a mouse model of skin infection. Fl-P(R)P(R)P(L)-5 showed potent activity against all clinical isolates of S. aureus tested, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA, respectively). Fl-P(R)P(R)P(L)-5 was also superior in clearing established in vitro biofilms of S. aureus and Staphylococcus epidermidis, compared with the established antimicrobials mupirocin and vancomycin. Additionally, topical treatment of an MRSA-infected wound with Fl-P(R)P(R)P(L)-5 enhanced wound closure and significantly reduced bacterial load. Finally, 0.5% Fl-P(R)P(R)P(L)-5 significantly reduced the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) in wounds induced by MRSA skin infection. In conclusion, the results of this study suggest the potential application of Fl-P(R)P(R)P(L)-5 in the treatment of staphylococcal skin infections.

Scanning fluorescence correlation spectroscopy as a versatile tool to measure static and dynamic properties of soft matter systems.

We present the formalism and experimental implementation of scanning fluorescence correlation spectroscopy (SFCS) for the measurements of soft matter system structure and dynamics. We relate the SFCS function Fourier transform to the system intermediate scattering function and demonstrate how SFCS can be combined with specific labelling to measure the desired statistical and kinetic features of the system. Using DNA as a model polymer, we demonstrate the application of SFCS to measure (1) the static structure factor of the system, (2) polymer end-to-end distance distribution, and (3) polymer segmental dynamics in dilute and in dense solutions. The measured DNA end-to-end distance distributions are close to Gaussian. Implementing SFCS we obtain reliable data on segmental mean-square displacement kinetics in dense solutions, where the static FCS approach fails because of dye photobleaching. For moderate concentrations in the semidilute regime (at ∼7 overlap concentrations) segmental dynamics exhibit only weak entanglements. Both of these experimental findings are consistent with theoretical predictions of the weakness of excluded interactions in semiflexible polymers.

Targeting intracellular bacteria with an extended cationic amphiphilic polyproline helix.

An extended cationic and amphiphilic polyproline helix (CAPH) is described with a dual mode of action: effective cell penetration of human macrophages, and potent antimicrobial activity in vitro against both Gram-positive and negative pathogens, including Acinetobacter baumannii, Escherichia coli O157 and Bacillus anthracis. This dual action was successfully combined to clear pathogenic bacteria (Brucella and Salmonella) residing within macrophages.

Structure of DNA coils in dilute and semidilute solutions.

We apply scanning fluorescence correlation spectroscopy to study the structure of individual DNA coils in dilute and semidilute solutions. In dilute solutions, over two decades in length, from 0.6 to 46 µm, DNA behave as ideal chains, in agreement with theoretical predictions and in disagreement with prior experiments. In semidilute solutions, up to very high densities, the structures of individual DNA coils are independent of concentration, unlike flexible coils that shrink with increasing density. Our experimental findings are consistent with the marginal solution theory of semiflexible polymers.

Hypernatremia in a patient treated with sodium polystyrene sulfonate.

Severe hyperkalemia requires urgent medical attention and correction in order to prevent arrhythmic complications. Sodium polystyrene sulfonate (SPS) is a cation exchange resin commonly used in the management of hyperkalemia. A recent review raised concerns regarding its effectiveness and potential adverse effects. Hypernatremia in adults in the setting of sodium polystyrene sulfonate therapy has not been described in the literature. We report the case of a woman who developed hypernatremia in the setting of excessive SPS administration and hope to increase awareness among clinicians regarding this potential side effect of SPS therapy.

Kidney injury molecule-1 expression identifies proximal tubular injury in urate nephropathy.

Urate nephropathy in children is uncommon, occurring mostly in those who have undergone chemotherapy or radiotherapy. The characteristic obstruction of distal nephron tubules by uric acid precipitates is considered key to the subsequent parenchymal injury. Whether proximal tubular injury plays a role in urate nephropathy remains unclear. We report one case of acute urate nephropathy and one case of chronic urate nephropathy in two pediatric patients. In their renal biopsies, we demonstrate upregulation of kidney injury molecule-1 (KIM-1), a specific injury marker, in proximal tubular cells. This finding implicates a role for proximal tubular injury in urate nephropathy.

Nephrotic range proteinuria: rare manifestation of scleroderma renal crisis.

The nephrotic range of proteinuria is uncommon in scleroderma renal crisis. This 46-yr-old woman with a medical history of scleroderma presented with very high blood pressure, a sudden elevation of serum creatinine, and proteinuria in the nephrotic range. Renal biopsy revealed onion-skin type of arterial changes with necrosis, confirming the presence of scleroderma nephropathy. Electron microscopy showed diffuse fusion of foot processes. Immunohistochemical staining (IHC) revealed increased expression in glomeruli of phosphorylated mammalian target of rapamycin (p-mTOR). These findings suggest that fusion of foot processes and activation of mammalian target of rapamycin-dependent pathways in podocytes are most likely responsible for the severe proteinuria in this patient with scleroderma nephropathy.