Cancer cells employ an evolutionarily conserved polyploidization program to resist therapy.

Unusually large cancer cells with abnormal nuclei have been documented in the cancer literature since 1858. For more than 100 years, they have been generally disregarded as irreversibly senescent or dying cells, too morphologically misshapen and chromatin too disorganized to be functional. Cell enlargement, accompanied by whole genome doubling or more, is observed across organisms, often associated with mitigation strategies against environmental change, severe stress, or the lack of nutrients. Our comparison of the mechanisms for polyploidization in other organisms and non-transformed tissues suggest that cancer cells draw from a conserved program for their survival, utilizing whole genome doubling and pausing proliferation to survive stress. These polyaneuploid cancer cells (PACCs) are the source of therapeutic resistance, responsible for cancer recurrence and, ultimately, cancer lethality.

An electric bottle for colloids.

Particle concentration is a dominant control parameter for colloids and other soft matter systems. We demonstrate a simple technique, "dielectrophoretic equilibrium," implemented as an "electric bottle," a planar capacitor in a larger volume. The uniform field in the capacitor traps particles in this force-free region at a higher density than in the zero field regions outside. We show how the technique measures the equation of state and we initiate and grow colloidal crystals. "Dielectrophoretic equilibria" enable the study of a complete concentration-dependent phase diagram from a single microscopic sample, obviating the previous need for preparing a large number of samples.

Insulating behavior of lambda-DNA on the micron scale.

We have investigated the electrical conductivity of lambda-DNA using DNA covalently bonded to Au electrodes. Thiol-modified dTTP was incorporated into the "sticky" ends of bacteriophage lambda-DNA using DNA polymerase. Two-probe measurements on such molecules provide a hard lower bound for the resistivity rho>10(6)Omega cm at bias potentials up to 20 V, in conflict with recent claims of moderate to high conductivity. By direct imaging, we show that the molecules are present after the measurements. We stress the importance of eliminating salt residues in these measurements.

Excited-state lifetimes of far-infrared collective modes in proteins.

Vibrational excitations of low frequency collective modes are essential for functionally important conformational transitions in proteins. Here we report the first direct measurement on the lifetime of vibrational excitations of the collective modes at 87 pm (115 cm(-1)) in bacteriorhodopsin, a transmembrane protein. The data show that these modes have extremely long lifetime of vibrational excitations, over 500 picoseconds, accommodating 1500vibrations. We suggest that there is a connection between this relativelyslow anharmonic relaxation rate of approximately 10 g sec(-1) and thesimilar observed rate of conformational transitions in proteins, which require require multi-level vibrational excitations and energy exchanges with othervibrational modes and collisional motions of solvent molecules.

Design of a microfabricated magnetic cell separator.

A new magnetic separation idea utilizing several ideas from microfabrication and nanomagnetics is presented. The basic idea comes from our earlier work using asymmetry in obstacles and Brownian motion to effect separation of objetcs [10] by moving them in streams whose angle to the hydrodynamic average velocity is a function of the diffusion coefficient of the object. The device we propose here is not technically a Brownian ratchet device but uses the idea of force which acts at angle to the hydrodynamic flow. In our case, the force is generated by a magnetic field gradient which comes from an array of magnetized wires which lie at an angle 0 to a hydrodynamic field flow. The sum of the hydrodynamic force and the magnetic force create a new vector which as in the case of the Brownian ratchet moves the cell out of the main stream direction.

Time resolved collapse of a folding protein observed with small angle x-ray scattering.

High-intensity, "pink" beam from an undulator was used in conjunction with microfabricated rapid-fluid mixing devices to monitor the early events in protein folding with time resolved small angle x-ray scattering. This Letter describes recent work on the protein bovine beta-lactoglobulin where collapse from an expanded to a compact set of states was directly observed on the millisecond time scale. The role of chain collapse, one of the initial stages of protein folding, is not currently understood. The characterization of transient, compact states is vital in assessing the validity of theories and models of the folding process.

Lifetimes of intermediates in the beta -sheet to alpha -helix transition of beta -lactoglobulin by using a diffusional IR mixer.

The extremely slow alpha-helix/beta-sheet transition of proteins is a crucial step in amylogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. These internal structural rearrangements within an already folded protein are a critical aspect of biological action and are a product of conformational flow along unknown metastable local minima of the energy landscape of the compact protein. We use a diffusional IR mixer with time-resolved Fourier transform IR spectroscopy capable of 400-micros time resolution to show that the trifluoroethanol driven beta-sheet to alpha-helix transition of beta-lactoglobulin proceeds via a compact beta-sheet intermediate with a lifetime of 7 ms, small compared with the overall folding time of beta-lactoglobulin.

The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin.

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O(2), and H(2)O(2) in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

Separation of 100-kilobase DNA molecules in 10 seconds.

Long double-stranded DNA molecules were separated in microfabricated hexagonal arrays in less than 1 min, several orders of magnitude faster than by using conventional technology. DNA samples were first concentrated at the entrance to the array in a thin band by entropic focusing. They were then separated by pulsed field electrophoresis. T4 (168.9 kbp) and lambda (48.5 kbp) DNAs could be resolved into two clearly separated bands in approximately 10 s in these experiments. This corresponds to a mass resolution of 6% in 11 min in a 1-cm-long array.

Long-lived amide I vibrational modes in myoglobin.

Pump-probe experiments in the infrared measure vibrational relaxation rates. Myoglobin, which is almost entirely alpha helix in secondary structure, has an unusually long, nonexponential excited state relaxation generated by optically pumping at the blue side ( 5. 85 microm) of the amide I band. The amino acid alanine and the predominantly beta sheet protein photoactive yellow protein do not have such a long-lived state, suggesting that the alpha helix in proteins can support nonlinear states of 15 ps characteristic times.

Sorting biomolecules with microdevices.

Micro- and nanofabrication techniques have provided an unprecedented opportunity to create a designed world in which separation and fractionation technologies which normally occur on the macroscopic scale can be optimized by designing structures which utilize the basic physics of the process, or new processes can be realized by building structures which normally do not exist without external design. Since microfabrication is exceedingly sophisticated in its development, it is possible to design and construct highly creative microdevices which allow one to probe specific aspects of biological objects. We give examples of uses of micro- and nanofabrication which, as opposed to simply shrinking the size of the vessels or tubes used in macroscopic lab environments, utilize our understanding of the physics of the process to take advantage of fabrication technologies.

Sorting by diffusion: an asymmetric obstacle course for continuous molecular separation.

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15-30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.

Effect of chronic m-CPP on locomotion, hypophagia, plasma corticosterone and 5-HT2C receptor levels in the rat.

1. The present study examined 5-HT2C receptor agonist-induced behavioural tolerance and 5-HT2C receptor down-regulation in adult rat brain. The effect of chronic subcutaneous infusion of the 5-HT2C receptor agonist, m-chlorophenylpiperazine (m-CPP, 10 mg kg(-1), day(-1)), for 14 days was examined on daily food intake, the ability of acute m-CPP (2.5 mg kg(-1), i.p.) to induce hypolocomotion in a novel arena and elevate plasma corticosterone levels and on ex vivo cortical [3H]-mesulergine binding and hippocampal 5-HT2C receptor protein levels. 2. Before chronic infusion, m-CPP (2.5 mg kg(-1), i.p.) attenuated the number of turns and rears made in a novel open field arena. In contrast, while m-CPP still elicited this hypolocomotion following 14 days, saline infusion, no such hypolocomotion occurred in rats given chronic m-CPP (10 mg kg(-1) day(-1)), indicating that almost complete tachyphylaxis of this behaviour occurred with chronic 5-HT2C receptor agonist injection. 3. During chronic infusion of m-CPP, rats consumed less food per day than saline-treated controls. Acute challenge with m-CPP following two weeks, treatment still attenuated food intake over the next four hours (by 43% and 30%, respectively from that on the previous day) in saline and m-CPP infusion groups, showing that only partial tolerance to 5-HT2C receptor agonist-induced hypophagia occurred. 4. In naive home cage rats, plasma corticosterone was elevated in a dose-dependent manner 35 min after m-CPP injection (0.5, 1 and 3 mg kg(-1), i.p.) but levels were comparable to control values 16 h after m-CPP (2, 5 and 10 mg kg(-1), i.p.). Sixteen hours after a single m-CPP injection (2.5 mg kg(-1), i.p.), plasma corticosterone levels were comparable in a group of rats which had received 14 days infusion of m-CPP or saline. However, following a similar acute m-CPP injection (2.5 mg kg(-1), i.p., - 16 h) in rats previously infused for 14 days with m-CPP, plasma corticosterone levels were lower than those in a separate group which received no chronic infusions (but only acute m-CPP injection), even though the plasma m-CPP levels were comparable in both groups. The data are consistent with the proposal that chronic m-CPP induced some down-regulation of hypothalamic 5-HT2C receptors which contribute, in a tonic manner, to plasma corticosterone secretion under the conditions investigated. 5. Chronic m-CPP infusion reduced the amount of [3H]-mesulergine binding (by 27%, without altering the KD) in membranes prepared from parietal/occipital/temporal cortex (under conditions to exclude binding to 5-HT2A receptors) and 5-HT2C receptor protein-like immunoreactive levels measured by radioimmunoassay in the hippocampus by 38%, confirming that 5-HT2C receptor down-regulation had occurred. 6. Even after 14 days m-CPP infusion only partial behavioural tolerance and 5-HT2C receptor down-regulation were observed, which may vary in different brain regions of the rat. Thus the hypophagia produced by m-CPP may involve activation of 5-HT2C receptors in the hypothalamus, where there is a greater receptor reserve or which are more resistant to agonist-induced down-regulation than 5-HT2C receptors in limbic areas (striatum and nucleus accumbens) mediating m-CPP-induced hypolocomotion.

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.

Sequencing in nanofabricated arrays: a feasibility study.

The feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing is investigated. A specific array design, consisting of rows of closely spaced posts, separated by longer open spaces, is proposed. Molecules driven through the array by an electric field get hooked over obstacles at successive rows and their progress through the device is delayed as a consequences. The dependence of the delay time on molecular size is derived. Numerical evaluation indicates that a device of modest dimensions, operating at high fields, can rapidly resolve oligonucleotides containing several hundred bases.

Biotechnology at low Reynolds numbers.

The shrinking of liquid handling systems to the micron and submicron size range entails moving into the area of small Reynolds numbers. The fluid dynamics in this regime are very different from the macroscale. We present an intuitive explanation of how the different physics of small Reynolds numbers flow, along with microscopic sizes, can influence device design, and give examples from our own work using fluid flow in microfabricated devices designed for biological processing.