Nanomechanical characterization by double-pass force-distance mapping

Cataloged from PDF version of article.We demonstrate high speed force–distance mapping using a double-pass scheme. The topography is measured in tapping mode in the first pass and this information is used in the second pass to move the tip over the sample. In the second pass, the cantilever dither signal is turned off and the sample is vibrated. Rapid (few kHz frequency) force–distance curves can be recorded with small peak interaction force, and can be processed into an image. Such a double-pass measurement eliminates the need for feedback during force–distance measurements. The method is demonstrated on self-assembled peptidic nanofibers

Interfiber interactions alter the stiffness of gels formed by supramolecular self-assembled nanofibers

Cataloged from PDF version of article.Molecular self-assembly is a powerful technique for developing novel nanostructures by using noncovalent interactions such as hydrogen bonding, hydrophobic, electrostatic, metal-ligand, p-p and van der Waals interactions. These interactions are highly dynamic and are often delicate due to their relatively weak nature. However, a sufficient number of these weak interactions can yield a stable assembly. In this work, we studied the mechanical properties of self-assembled peptide amphiphile nanostructures in the nanometre and micrometre scale. Hydrogen bonding, hydrophobic and electrostatic interactions promote self-assembly of peptide amphiphile molecules into nanofibers. Bundles of nanofibers form a three-dimensional network resulting in gel formation. The effect of the nanofiber network on the mechanical properties of the gels was analyzed by AFM, rheology and CD. Concentration and temperature dependent measurements of gel stiffness suggest that the mechanical properties of the gels are determined by a number of factors including the interfiber interactions and mechanical properties of individual nanofibers. We point out that the divergence in gel stiffness may arise from the difference in strength of interfiber bonds based on an energetic model of elastic rod networks, along with continuum mechanical models of bundles of rods. This finding differs from the results observed with traditional polymeric materials. Understanding the mechanisms behind the viscoelastic properties of the gels formed by self-assembling molecules can lead to development of new materials with controlled stiffness. Tissue engineering applications can especially benefit from these materials, where the mechanical properties of the extracellular matrix are crucial for cell fate determination. © The Royal Society of Chemistry 2011

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Mechanical forces in plant tissue matrix orient cell divisions via microtubule stabilization.

Plant morphogenesis relies exclusively on oriented cell expansion and division. Nonetheless, the mechanism(s) determining division plane orientation remain elusive. Here, we studied tissue healing after laser-assisted wounding in roots of Arabidopsis thaliana and uncovered how mechanical forces stabilize and reorient the microtubule cytoskeleton for the orientation of cell division. We identified that root tissue functions as an interconnected cell matrix, with a radial gradient of tissue extendibility causing predictable tissue deformation after wounding. This deformation causes instant redirection of expansion in the surrounding cells and reorientation of microtubule arrays, ultimately predicting cell division orientation. Microtubules are destabilized under low tension, whereas stretching of cells, either through wounding or external aspiration, immediately induces their polymerization. The higher microtubule abundance in the stretched cell parts leads to the reorientation of microtubule arrays and, ultimately, informs cell division planes. This provides a long-sought mechanism for flexible re-arrangement of cell divisions by mechanical forces for tissue reconstruction and plant architecture

An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface.

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization

Bioactive self-assembled peptide nanofibers for corneal stroma

Cataloged from PDF version of article.Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration