Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.protein transduction domain | polyarginine | peptide-lipid interactions | pore-forming peptide | antimicrobial peptide C ell-penetrating peptides (CPPs) are effective intracellular delivery systems (1-5). These peptides are usually short (<20 amino acids) and cationic. Examples include the TAT peptide from HIV, antennapedia (ANTP) from Drosophila, and even simple polyarginines. Although unique molecular architectures incorporating CPPs have been designed for drug delivery (3, 6-8), the molecular mechanisms of cellular entry, and the relations between them, are not well understood. Different uptake mechanisms have been proposed for CPPs (9). Cell-based assays have indicated that multiple endocytotic pathways are involved (10-15). In addition to these, CPPs are also capable of direct entry mechanisms* (17-20). In general, cell-penetrating activity of CPPs has proven to be difficult to eliminate completely using a specific set of conditions (3,12,21), suggesting the existence of multiple mechanisms. A unified understanding of CPPs, which is currently lacking, must engage why the same sequence can readily activate the qualitatively distinct outcomes.How do relatively simple molecules like HIV TAT peptide facilitate mechanisms as different as direct translocation, and multiple endocytotic processes? Rather than debate priority between mechanisms, we focus on the physical chemistry of what these different mechanisms and CPPs have in common. Here, we show how the TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different condition...

Polymeric vesicles are a relatively new class of nanoscale self-assembled materials that show great promise as robust encapsulants. Compared with liposomes, use of polymeric building blocks for membrane formation allows increased stability, stimuli responsiveness and chemical diversity, which may prove advantageous for drug-delivery applications . A major drawback of most polymeric vesicles is the lack of biofunctionality, which restricts their ability to interact with cells and tissues. We have prepared vesicles composed of polyarginine and polyleucine segments that are stable in media, can entrap water soluble species, and can be processed to different sizes and prepared in large quantities. The remarkable feature of these materials is that the polyarginine segments both direct structure for vesicle formation and provide functionality for efficient intracellular delivery of the vesicles. This unique synergy between nanoscale self-assembly and inherent peptide functionality provides a new approach for design of multifunctional materials for drug delivery.

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.

In this paper, the features of the intensity-based Doppler variance (IBDV) method were analyzed systemically with a flow phantom. The effects of beam scanning density, flow rate and the time interval between neighboring A-lines on the performance of this method were investigated. The IBDV method can be used to quantify the flow rate and its sensitivity can be improved by increasing the time interval between the neighboring A-lines. A higher sensitivity IBDV method that applies the algorithm along the slower scan direction was proposed. In comparison to laser speckle imaging maps of blood flow, we demonstrated the ability of the method to identify vessels with altered blood flow. In clinical measurements, we demonstrated the ability of the method to image vascular networks with exquisite spatial resolution and at depths up to 1.2 mm in human skin. These results collectively demonstrated the potential of the method to monitor the microvasculature during disease progression and in response to therapeutic intervention.