Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering.

Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.

Light-activated porphyrin-based formulations to inactivate bacterial spores.

AIM: The objective of this study was to develop porphyrin-based formulations to inactivate Bacillus spores. We probed the effect of porphyrins alone and in combination with germinants against both Bacillus cereus and Bacillus anthracis spores in the presence of light. METHODS AND RESULTS: We tested the effect of two different porphyrins, amine-modified protoporphyrin IX (PPIX) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Treatment with the porphyrins alone did not significantly influence spore viability. However, when spores were pretreated with a solution containing the germinants, l-alanine and inosine, the spore viability dropped by as much as 4.5 logs in the presence of light. The extent of inactivation depended on the germination conditions and the type of porphyrin used, with TMP being more effective. CONCLUSION: Porphyrins can be used effectively in combination with germinants to inactivate Bacillus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide evidence that porphyrins can be used to inactivate Bacillus spores in the presence of germinants and light irradiation. This finding may be general and may be extended to spores of other pathogens.

Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery.

Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

Selective assembly of multi-component nanosprings and nanorods.

This communication proposes a new approach to create complex hierarchical nano-to-meso-scale architectures based on the use of biological connector molecules to direct the assembly of uniquely shaped multi-component nanostructures fabricated using glancing angle deposition (GLAD). Multiple sets of 50-nm-wide and 150 to 650-nm-tall Si-Cr/Au multi-stack zigzag nanosprings and nanorods are grown by GLAD on Si substrates. Nanorods, chosen for selective assembly, are detached from the substrate, suspended in an aqueous solution, and their surfaces are selectively functionalized by attaching biotin and streptavidin connector-molecules to the Au-regions. Successive mixing of different suspensions leads to the end-to-end assembly of long and short nanorods. This technique provides the path to build hybrid nano-architectures including nano-honeycombs, nanoladders, and 3D nanorod networks, comprised of controlled material combinations.

Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides.

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

Effect of filler geometry on interfacial friction damping in polymer nanocomposites.

Single-walled carbon nanotube polycarbonate and C60 polycarbonate nanocomposites were fabricated using a solution mixing method. The composite loss modulus was characterized by application of dynamic (sinusoidal) load to the nanocomposite and the pure polymer samples. For a loading of 1 weight %, the single-walled nanotube fillers generated more than a 250% increase in loss modulus compared to the baseline (pure) polycarbonate. Even though the surface area to volume ratio and surface chemistry of C60 is similar to that for nanotubes, we report no significant increase in the energy dissipation for the 1% weight C60 nanoparticle composite compared to the pure polymer. We explain these observations by comparing qualitatively, the active sliding area (considering both normal and shear stresses) for a representative volume element of the nanotube and the nanoparticle composites. These results highlight the important role played by the filler geometry in controlling energy dissipation in nanocomposite materials.

Designing a polyvalent inhibitor of anthrax toxin.

Screening peptide libraries is a proven strategy for identifying inhibitors of protein-ligand interactions. Compounds identified in these screens often bind to their targets with low affinities. When the target protein is present at a high density on the surface of cells or other biological surfaces, it is sometimes possible to increase the biological activity of a weakly binding ligand by presenting multiple copies of it on the same molecule. We isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. A molecule consisting of multiple copies of this nonnatural peptide, covalently linked to a flexible backbone, prevented assembly of the toxin complex in vitro and blocked toxin action in an animal model. This result demonstrates that protein-protein interactions can be inhibited by a synthetic, polymeric, polyvalent inhibitor in vivo.

Basicity of the amino groups of the aminoglycoside amikacin using capillary electrophoresis and coupled CE-MS-MS techniques.

This paper describes the use of capillary electrophoresis (CE), and coupled CE and mass spectrometric techniques, to measure the values of the pKa of the amino groups of the aminoglycoside antibiotic amikacin and of its acetylated derivatives. These values of pKa (8.4, 6.7, 9.7, 8.4) were determined by measuring the electrophoretic mobilities of the molecules as a function of pH; they are within 0.7 unit of certain values reported in the literature (by 13C and 15N NMR spectroscopies) but resolved ambiguities left by these earlier studies. The range of values of pKa of amino groups also indicates the complex dependence of the acidity of a functional group (and thus the extent of ionization at a specified value of pH) on the molecular environment of that group.

Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems.

Three-dimensional microfluidic systems were fabricated and used to pattern proteins and mammalian cells on a planar substrate. The three-dimensional topology of the microfluidic network in the stamp makes this technique a versatile one with which to pattern multiple types of proteins and cells in complex, discontinuous structures on a surface. The channel structure, formed by the stamp when it is in contact with the surface of the substrate, limits migration and growth of cells in the channels. With the channel structure in contact with the surface, the cells stop dividing once they form a confluent layer. Removal of the stamp permits the cells to spread and divide.

Patterning proteins and cells using soft lithography.

This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

Cardiopulmonary resuscitation policies in long-term care facilities.

OBJECTIVES: To describe CPR policies and the procedures for discussing CPR policies of Wisconsin long-term care facilities. DESIGN: Mail survey and telephone interview. MEASUREMENTS: Information about CPR policy, how policy is disclosed to residents and by whom, emergency medical technician team (EMT) response time, and number of CPR attempts during 1993. RESULTS: The 1994 survey response rate was 85% (346/ 404 facilities). Four percent of responding facilities maintain a policy of never initiating CPR. Another 23% never initiate CPR but would call an EMT. Lack of efficacy was the usual basis for policies never initiating CPR. About 15% of facilities would initiate CPR only on residents who had previously indicated a preference. On individuals who had not made an advanced directive decision, 57% of facilities would initiate CPR in the event of an arrest. Almost 30% of facilities offering CPR would initiate CPR on unwitnessed arrests. Approximately 51% of all facilities assigned a social worker alone to discuss CPR policy and preference, whereas 12.5% assigned a physician alone or as part of a team. During 1993, an estimated 118 attempts at CPR were reported for 172 facilities with a total of 19,596 licensed beds, for a frequency of one CPR attempt per 166 beds per year. CONCLUSIONS: Poor efficacy in this population was the main reason given for policies of never initiating CPR. Specific factors relating to CPR efficacy, such as EMT response time and ease of maintaining trained staff, were not major influences. Almost 30% of facilities offering CPR would perform it in unwitnessed situations, despite unlikely success. Many decisions about CPR may not be fully informed as nurses and physicians are not often assigned to discuss advance directives with residents or surrogates. Utilization of CPR in nursing homes offering resuscitation is low.

Minimal trauma fractures in older nursing home residents: the interaction of functional status, trauma, and site of fracture.

OBJECTIVE: This study was conducted to determine the incidence of long bone fractures in institutionalized older persons and to describe preceding traumatic events and the functional status of individuals sustaining fractures. DESIGN: A 1-year, prospective, cumulative incidence survey. SETTING: Eleven skilled nursing care facilities in the state of Wisconsin. PATIENTS: All residents of the 11 facilities. MEASUREMENTS: All incident reports of long bone fractures, description of events preceding the fractures, and functional status of the fracture cases. In addition, demographic and medical information was collected on fracture cases and the general nursing home population. MAIN RESULTS: Overall long bone fracture incidence was 3.52 per 100 subjects per year. Minimal trauma fracture incidence was 0.84 per 100 subjects per year. Fracture location was significantly related to type of trauma. Functional status was significantly related to fracture location and to the type of trauma preceding the fracture. Minimal trauma fractures occurred in individuals who were less mobile and more likely to be bed-bound, and the location was more likely to be the lower extremity below the hip. CONCLUSION: This is the first prospective survey of long bone and spontaneous fracture incidence rates in multiple nursing home facilities. Minimal trauma fractures are common in the nursing home, and most have no clear precipitating factors other than severely impaired mobility.

Factors affecting physician participation in nursing home care.

Physicians generally consider nursing home practice a low priority compared with other aspects of their practices. In order to encourage more enthusiastic physician involvement, negative influences needed to be identified so that corrective ideas can be formulated. Negative factors include low reimbursement, frequent office interruptions, excessive paperwork, and a sense of loss of authority. The problem of quality physician involvement must be viewed from new perspectives. An increase in reimbursement is only part of the solution. Other measures that enhance the professional image and responsibility will increase physician participation more than laws and regulations, which increase physician time commitment without increasing reimbursement. Laws and regulations, through misperception, misinterpretation, and misapplication, can have unintended adverse results. Only creative solutions addressing identified negative factors will cut through the Gordian knot, which prevents enthusiastic and quality medical care for all NH residents.