Carlo D. Montemagno

Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores.

Biological pores have been used to study the transport of DNA and other molecules but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter and exit during a viral infection, contains a connector protein that has a 3.6 – 6.0 nm wide channel. Here we show that a modified version of the connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, can act as conductive channels to allow the translocation of double-stranded DNA. Single-channel conductance assays and quantitative PCR confirmed the translocation through the pore. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have interesting applications in nanotechnology and nanomedicine, such as MEMS sensing, microreactors, gene delivery, drug loading, and DNA sequencing.

Nanotechnology in ophthalmology.

Nanotechnology involves the creation and use of materials and devices at the size scale of intracellular structures and molecules, and involves systems and constructs in the order of <100 nm. The aim of nanomedicine is the comprehensive monitoring, control, construction, repair, defence, and improvement of human biological systems at the molecular level, using engineered nanodevices and nanostructures that operate massively in parallel at the single-cell level, ultimately to achieve medical benefit. In this review we consider general principles of nanotechnology as applied to nanomedicine (e.g., biomimicry and pseudointelligence). Some applications of nanotechnology to ophthalmology are described (including treatment of oxidative stress; measurement of intraocular pressure; theragnostics; use of nanoparticles to treat choroidal new vessels, prevent scarring after glaucoma surgery, and treat retinal degenerative disease with gene therapy; prosthetics; and regenerative nanomedicine). Nanotechnology will revolutionize our approach to current therapeutic challenges (e.g., drug delivery, postoperative scarring) and will enable us to address currently unsolvable problems (e.g., sight-restoring therapy for patients with retinal degenerative disease). Obstacles to the incorporation of nanotechnology remain, such as safe manufacturing techniques and unintended biological consequences of nanomaterial use. These obstacles are not insurmountable, and revolutionary treatments for ophthalmic diseases are expected to result from this burgeoning field.

Volumetric imaging of aperture distributions in connected fracture networks

We use x-ray computerized tomographic (CT) imaging to present quantitative aperture data for three-dimensional interconnected fracture networks imbedded in intact opaque rock samples several centimeters in length. X-ray images are obtained by injecting a high-density liquid metal into the fractured rock specimen under lithostatic conditions. The combination of tomographic reconstruction with gravimetric analysis makes it possible for the first time to obtain effective fracture aperture sizes to an accuracy of only several microns, located spatially within 300 microns. The apertures in the fracture network are spatially correlated over distances of 10 mm to 30 mm. The apertures of the intersections of the fractures were not found to be statistically larger in size than for the complete fracture network

One-way traffic of a viral motor channel for double-stranded DNA translocation.

Linear double-stranded DNA (dsDNA) viruses package their genome into a procapsid using an ATP-driven nanomotor. Here we report that bacteriophage phi29 DNA packaging motor exercises a one-way traffic property for dsDNA translocation from N-terminal entrance to C-terminal exit with a valve mechanism in DNA packaging, as demonstrated by voltage ramping, electrode polarity switching, and sedimentation force assessment. Without the use of gating control as found in other biological channels, the observed single direction dsDNA transportation provides a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or high throughput DNA sequencing apparatus.

Metabolism of 2,4,6-trinitrotoluene by aPseudomonas consortium under aerobic conditions

An aerobic bacterial consortium was shown to degrade 2,4,6-trinitrotoluene (TNT). At an initial concentration of 100 ppm, 100% of the TNT was transformed to intermediates in 108 h. Radiolabeling studies indicated that 8% of [14C]TNT was used as biomass and 3.1% of [14C]TNT was mineralized. The first intermediates observed were 4-amino-2,6-dinitrotoluene and its isomer 2-amino-4,6-dinitrotoluene. Prolonged incubation revealed signs of ring cleavage. Succinate or another substrate—e.g., malic acid, acetate, citrate, molasses, sucrose, or glucose—must be added to the culture medium for the degradation of TNT. The bacterial consortium was composed of variousPseudomonas spp. The results suggest that the degradation of TNT is accomplished by co-metabolism and that succinate serves as the carbon and energy source for the growth of the consortium. The results also suggest that this soil bacterial consortium may be useful for the decontamination of environmental sites contaminated with TNT.

Protein-driven energy transduction across polymeric biomembranes

Block copolymer-based membrane technology enables the development of a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. These active materials possess a broad applicability in areas such as the enhancement of existing technologies or production of current-generating films for power sources. For example, these active materials can be integrated with fuel cell ion transport membranes such as Nafion® in order to improve the ability of Nafion® to retain leaking protons. Also, the demonstration of protein-driven current production across these membranes represents a possible alternative power source that is both highly efficient and light in weight. Our work has demonstrated the fabrication of large-area copolymer biomembranes that are functionalized by bacteriorhodopsin (BR) and cytochrome c oxidase (COX) ion transport proteins. Among their many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods such as Langmuir–Blodgett (LB) deposition. Following the large-scale insertion of proteins into block copolymer LB films, we have demonstrated significant pH changes based upon light-actuated proton pumping. Protein activity across the BR and COX-functionalized membrane has also been observed using impedance spectroscopy as well as direct current measurement.

Measurement of fluid contents by light transmission in transient three-phase oil-water-air systems in sand

Most three-phase flow models lack rigorous validation because very few methods exist that can measure transient fluid contents of the order of seconds of whole flow fields. The objective of this study was to develop a method by which fluid content can be measured rapidly in three-phase systems. The method uses the hue and intensity of light transmitted through a slab chamber to measure fluid contents. The water is colored blue with CuSO4. The light transmitted by high-frequency light bulbs is recorded with a color video camera in red, green, and blue and then converted to hue, saturation, and intensity. Calibration of hue and intensity with water, oil, and air is made using cells filled with different combinations of the three fluids. The results show that hue and water content are uniquely related over a large range of fluid contents. Total liquid content is a function of both hue and light intensity. The air content is obtained by subtracting the liquid content from the porosity. The method was tested with static and transient experiments. Measurements made with the light transmission method (LTM) and synchrotron X rays of the static experiment agreed well. In the transient experiments, fingers were formed by dripping water on the surface in a two-dimensional slab chamber with partially oil-saturated sand. The LTM is able to capture the spatial resolution of the fluid contents and can provide new insights in rapidly changing, three-phase flow systems.

Artificial Photosynthesis in Ranaspumin-2 Based Foam

We present a cell-free artificial photosynthesis platform that couples the requisite enzymes of the Calvin cycle with a nanoscale photophosphorylation system engineered into a foam architecture using the Tungara frog surfactant protein Ranaspumin-2. This unique protein surfactant allowed lipid vesicles and coupled enzyme activity to be concentrated to the microscale Plateau channels of the foam, directing photoderived chemical energy to the singular purpose of carbon fixation and sugar synthesis, with chemical conversion efficiencies approaching 96%.

Biological transformation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT-contaminated soil

Abstract Four Pseudomonas spp. were isolated from a soil consortium enriched from soil contaminated with 2,4,6-trinitrotoluene (TNT). All four species extensively transformed TNT. The rate of transformation varied among species. In isolate 4, 100% of TNT (100 ppm) was transformed in 4 days. The TNT transformation was achieved by the four isolates through a co-metabolic process with a succinate co-substrate. The four isolates produced NO 2 − from TNT. The maximum NO 2 − production, observed for isolate 1, was equal to 30% of the NO 2 − available from the nitro groups of TNT. For other isolates the NO 2 − production varied from 10 to 16%. The radiolabeling studies showed signs ring cleavage. Isolate 3 used 13% of 14 C-TNT to make cellular material, and isolate 4 converted 6% of 14 C-TNT to biomass. The production of 14 C-CO 2 was observed for all four isolates, but the amount of 14 C-CO 2 produced was quite low: isolate 4 produced 14 C-CO 2 from approximately 1% of 14 C-TNT. The rate of degradation of TNT intermediates was very slow, reflecting possible difficulties in metabolizing the intermediates of TNT to CO 2 . The main intermediates were identified as 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene.

Source water assessment and nonpoint sources of acutely toxic contaminants: A review of research related to survival and transport of Cryptosporidium parvum

Amendments to the Safe Drinking Water Act (PL-930123) in 1996 required that public water supply managers identify potential sources of contamination within contributing areas. Nonpoint sources of acutely toxic microbial contaminants, such as Cryptosporidium parvum, challenge current approaches to source identification and management as a first step toward developing management plans for public water supply protection. Little may be known about survival and transport in the field environment, prescribed practices may not be designed to manage such substances, and infective stages may be present in vast numbers and may resist water treatment and disinfection processes. This review summarizes research related to survival and transport of C. parvum oocysts, as an example of an acutely toxic contaminant with nonpoint sources in animal agriculture. It discusses ∥1) significance of infected domesticated animals as potential sources of C. parvum, (2) laboratory and field studies of survival and transport, and (3) approaches to source control in the context of public health protection.