Experimental study of diffusion-based extraction from a cell suspension

A recently proposed application of microfluidics is the post-thaw processing of biological cells. Numerical simulations suggest that diffusion-based extraction of the cryoprotective agent dimethyl sulfoxide (DMSO) from blood cells is viable and more efficient than centrifugation, the conventional method of DMSO removal. In order to validate the theoretical model used in these simulations, a prototype was built and the flow of two parallel streams, a suspension of Jurkat cells containing DMSO and a wash stream that contained neither cells nor DMSO, was characterized experimentally. DMSO transport in a rectangular channel (depth 500 μm, width 25 mm and overall length 125 mm) was studied as a function of three dimensionless parameters: depth ratio of the streams, cell volume fraction in the cell solution, and the Peclet number (Pe) based on channel depth, average flow rate and the diffusion coefficient for DMSO in water. In our studies, values of Pe ranged from O(10³) to O(10⁴). Laminar flow was ensured by keeping the Reynolds number between O(1) and O(10). Experimental results based on visual and quantitative data demonstrate conclusively that a microfluidic device can effectively remove DMSO from liquid and cell laden streams without compromising cell recovery. Also, flow conditions in the microfluidic device appear to have no adverse effect on cell viability at the outlet. Further, the results demonstrate that we can predict the amount of DMSO removed from a given device with the theoretical model mentioned previously.     

Predictability of healing of ischemic leg ulcers by radioisotopic and Doppler ultrasonic examination

In a prospective study of twenty-six patients with ischemic ulcerations of the lower extremity, the predictive reliability with regard to spontaneous wound healing of diabetes, pedal pulses, ankle blood pressure (ABP) as measured by doppler ultrasound, and "leg ulcer scan" as performed by the intra-arterial injection of radioactive albumin was evaluated. The results suggest that only the leg ulcer scan is significantly reliable in predicting the likelihood of spontaneous healing. The following format for the evaluation of the ischemic leg ulcer is therefore suggested: (1) If pedal pulses are present, a three week trial of conservative therapy is indicated before further evaluation. (2) If the doppler ABP is 50 mm Hg or less, the ulcer will not heal spontaneously. (3) Leg ulcer scan is indicated: (a) in the absence of pedal pulses if ABP is less than 50 mm Hg; (b) in the presence of pedal pulses if there is no evidence of spontaneous healing after three weeks of conservative therapy.     

Energetically optimal running requires torques about the centre of mass

Bipedal animals experience ground reaction forces (GRFs) that pass close to the centre of mass (CoM) throughout stance, first decelerating the body, then re-accelerating it during the second half of stance. This results in fluctuations in kinetic energy, requiring mechanical work from the muscles. However, here we show analytically that, in extreme cases (with a very large body pitch moment of inertia), continuous alignment of the GRF through the CoM requires greater mechanical work than a maintained vertical force; we show numerically that GRFs passing between CoM and vertical throughout stance are energetically favourable under realistic conditions; and demonstrate that the magnitude, if not the precise form, of actual CoM-torque profiles in running is broadly consistent with simple mechanical work minimization for humans with appropriate pitch moment of inertia. While the potential energetic savings of CoM-torque support strategies are small (a few per cent) over the range of human running, their importance increases dramatically at high speeds and stance angles. Fast, compliant runners or hoppers would benefit considerably from GRFs more vertical than the zero-CoM-torque strategy, especially with bodies of high pitch moment of inertia—suggesting a novel advantage to kangaroos of their peculiar long-head/long-tail structure.     

Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active beta subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.     

Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.     

Current and future therapies for HCV infection: what should the end point for treatment be?

Based upon all of the available data relating to the natural history, chemical course, and response to therapy of HCV, the following recommendations are made: 1) The primary end point for HCV therapy should be HCV clearance from all tissue sites, eg plasma, liver and others 2) Therapy should be provided for patients with early infections as they have the best chance of achieving a virologic response 3) Therapy should be offered to patients with cirrhotic disease, as prevention of hepatic decompensation and degeneration to hepatic cancer is possible 4) End stage decompensated disease should be treated, particularly if liver transplantation is being considered, in an effort to either eliminate or ameliorate disease recurrence 5) Combination therapies are preferable to monotherapy as they enhance the likelihood of a therapeutic response. Some of these include agents that reduce the frequency of IFN-induced untoward events (NSAIDs) 6) The approach to HCV infection should be to view it as an infectious disease. In this way, multi-agent therapy could be used to prevent the emergence of drug resistant mutants as well as to obtain earlier clearance of the infection than is possible with monotherapy.     

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.